Improved muconic acid production from genetically engineered microorganisms

ABSTRACT

The subject of this invention is improvements in the yield and titer of biological production of muconic acid by fermentation. Increased activity of one or more enzymes involved in the muconic acid pathway leads to increased production of muconic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of InternationalApplication No. PCT/US2017/020263 filed Mar. 1, 2017, which claimspriority to U.S. Provisional Patent Application No. 62/302,558 filedMar. 2, 2016.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 20, 2020, is named524823US_ST25.txt and is 344 kb in size.

FIELD OF THE INVENTION

The present invention is in the field of producing renewable chemicalfeedstocks using biocatalysts that have been genetically engineered inthe central aromatic biosynthetic pathway. More specifically, thepresent invention provides processes for improving the production ofmuconic acid :from renewable carbon resources using genetically modifiedbiocatalysts.

BACKGROUND OF THE INVENTION

Adipic acid is a major commodity chemical and is used in the productionof nylon 6,6 and polyurethanes. Adipic acid is currently derived frompetrochemical feedstocks. Current synthesis of adipic acid isenvironmentally harmful releasing nitrous acid (Xie et al., 2014).Alternatively, adipic acid can be made from any of the three isomers ofmuconic acid (cis, cis; cis, trans; trans, trans isomers) by chemicalhydrogenation. It would be desirable to produce muconic acid fromrenewable resources by fermentation with a microorganism, followed byhydrogenation process to yield adipic acid, since such a route to adipicacid would be more environmentally friendly than the traditionalpetrochemical route (Niu, Draths and Frost, 2002; Frost and Draths,1997). Many other chemicals can be made by chemical conversion of one ormore muconic acid isomers, including, but not limited to 1.6 hexanediol, 3-hexenedicarboxylic acid, 1,6-hexanediamine, and terephthalicacid.

The international patent application publication No. WO 2011/017560claims biocatalysts having a muconate pathway and a method for producingmuconic acid using these biocatalysts. In brief, this published patentapplication discloses four different pathways for producing muconicacid. The first pathway for muconic acid production starts withsuccinyl-CoA and acetyl-CoA. The second pathway for muconic acidproduction begins with pyruvate and malonate semialdehyde. The thirdpathway for muconic acid production starts with pyruvate and succinicsemialdehyde. The fourth pathway for muconic acid production starts withlysine. All these pathways for muconic acid production proposed in thispatent application are based on computer modeling and it is yet to beseen whether such biocatalysts can ever be created with commerciallyacceptable productivity and yield for muconic acid.

A fermentation route to produce cis, cis-muconic acid using agenetically engineered E. coli system has been described in thescientific literature (Niu et al., 2002; Frost and Draths, 1997) and inpatent literature (U.S. Pat. Nos. 5,487,987; 5,616,496; WO 2011/085311A1). However, the prior art process for muconic acid production sufferedfrom significant drawbacks such as expensive medium components (aromaticamino acids and vitamins) and chemical inducers, as well as yields lowerthan required for industrial production. A recent United States PatentApplication Publication No. US2015/0044755, which is incorporated hereinby reference in its entirety, provides improved biocatalysts for muconicacid production involving constitutive expression of relevant gene,improved heterologous genes and novel “leaky” AroE enzymes. The presentinvention provides further improvements in the muconic acid biocatalystsdescribed in the United States Patent Application Publication No.US2015/0044755.

SUMMARY OF THE INVENTION

This present invention provides genetically engineered microorganismsthat produce cis, cis-muconic acid starting from non-aromatic carbonsources, such as sugars and carbohydrates including, but not limited toglucose, sucrose, glycerol and cellulosic hydrolysate.

In one embodiment of the present invention, the activity of a negativeregulator of aromatic amino acid biosynthesis is geneticallymanipulated. In one aspect of the present invention, the activity of thenegative regulator TyrR is substantially reduced by means of controllingthe expression of the tyrR gene coding for the TyrR protein. In anotheraspect of the present invention, the activity of the negative regulatorTyrR is totally eliminated by means of deleting or inactivating the tyrRgene in the chromosomal DNA of the microorganism.

In another embodiment of the present invention, the feedback inhibitionof certain enzymes in the aromatic amino acid pathway by certainmetabolites is overcome through genetic manipulations. In most wild typeE. coli strains, deoxyarabino-heptulosonate 7-phosphate synthase (“DAHPsynthase”) functioning at the beginning of the aromatic amino acidpathway occurs as three different isozymes which are known to be encodedby three different genes namely aroG, aroF and aroH. The proteinsencoded by each of these three genes are subjected to feedbackinhibition by one or more metabolites of aromatic amino acid pathway. Inone aspect of the present invention, the wild type aroG gene is replacedby a modified aroG gene which codes for an AroG protein that isresistant to feedback inhibition by one or more metabolites of thearomatic amino acid pathway within the microbial cell. Such a feedbackresistant form of AroG protein is referred as “AroG^(FBR)”. In anotheraspect of the present invention, the wild type aroF gene is replaced byan aroF gene which codes for an AroF protein that is resistant tofeedback inhibition by one or more metabolites of the aromatic aminoacid pathway within the microbial cell (AroF^(FBR)). In yet anotheraspect of the present invention, the wild type aroH gene is replaced byan aroH gene which codes for an AroH protein that is resistant tofeedback inhibition by one or more metabolites of the aromatic aminoacid pathway within the microbial cell (AroH^(FBR)). In yet anotheraspect of the present invention the biocatalyst selected for thecommercial production of cis, cis-muconic acid may have more than onefeedback resistant isozyme for DAHP synthase.

In another embodiment of the present invention, the activity of one ormore of the enzymes involved in the central aromatic biosyntheticpathway within the microbial cell is enhanced. In one aspect of thepresent invention, the enhancement of the activity of one or moreenzymes involved in the operation of an aromatic pathway and/or amuconic acid pathway is accomplished through genetic manipulation. In apreferred aspect of the present invention, the expression of one or moreof the genes coding for the proteins AroF, AroG, AroH, AroB, TktA, TalB,AroZ, QutC, Qa-4, AsbF, QuiC, AroY, Rpe, Rpi, Pps, CatA and CatX ortheir homologs or analogs are enhanced leading to the increased activityof said proteins. Rpe is a ribulose-5-phosphate epimerase, Rpi is aribulose-5-phosphate isomerase, and Pps is a phosphoenol pyruvatesynthetase (Neidhardt and Curtiss, 1996). If the host strain is yeast,for example Saccharomyces cerevisiae, or a filamentous fungus, forexample, Neurospora crassa, several of the enzymes that catalyzereactions in the shikimate pathway can be combined into one largeprotein or polypeptide, called Aro1p, encoded by the ARO1 gene in thecase of S. cerevisiae. Aro1p combines the functions of AroB, AroD, AroE,AroK (or AroL), and AroA). As such, for the purposes of this invention,Aro1p, and ARO1, or a portion thereof, can be used as a substitute, orin addition to, AroB, AroD, AroE, AroK, and/or AroA.

In yet another embodiment of the present invention, flux througherythrose-4-phosphate within the bacterial cell is enhanced by means ofoverexpressing enzymes in the operation of the pentose phosphatepathway. In one aspect of the present invention, the expression oftransaldolase enzyme, for example one coded by the talB or talA gene isenhanced by genetic modification. In another aspect of the presentinvention, the expression of a gene encoding transketolase enzyme, forexample, the tktA gene is enhanced by genetic manipulations. In yetanother aspect of the present invention, the expression of the genesencoding either or both ribulose-5-phosphate epimerase andribulose-5-phosphate isomerase are enhanced by genetic manipulations.

In another embodiment of the present invention, the pool of thephosphoenol pyruvate (PEP) necessary for the functioning of the aromaticamino acid pathway is increased through genetic manipulation. In oneaspect of the present invention, competition for the use of PEP pool isdecreased through elimination and/or complementation of a PEP-dependentphosphotransferase system (PTS) for glucose uptake with a PEPindependent system for glucose uptake. In another aspect of the presentinvention, the GalP based sugar uptake system is inactivated for thepurpose of conserving ATP within the microbial cells. In yet anotheraspect of the present invention, in the microbial cells defective in thefunctioning of both PTS system and GalP based sugar uptake system(ΔPTS/ΔgalP), the sugar uptake is accomplished by means of introducingan exogenous gene encoding for Glf (protein facilitating the glucosediffusion), or exogenous genes encoding for both Glf and Glk(glucokinase) proteins. In yet another embodiment of the presentinvention, the availability of PEP is increased by increasing theexpression of a gene that encodes a PEP synthetase, such as pps.

In yet another embodiment of the present invention, the activity of3,4-dihydroxybenzoic acid decarboxylase (AroY) is enhanced. In oneaspect of this embodiment, the expression of AroY is enhanced by geneticmanipulation. In another aspect of the present invention, the expressionof a protein acting as an accessory protein to AroY, selected from agroup comprising UbiX, KpdB, Elw, Kox, Lpl and homologs thereof, isincreased by genetic manipulation leading to an increase in3,4-dihydroxybenzoic acid decarboxylase activity.

In another embodiment of the present invention, PEP availability isincreased by the reduction or elimination of phosphoenolpyruvatecarboxylase (Ppc) activity. In one aspect of the present invention,pyruvate carboxylase (Pyc) activity is increased and/or substituted forPpc activity, particularly when Ppc activity is eliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Pathway for aromatic amino acid biosynthesis in E. coli.

FIG. 2. Pathway for muconic acid biosynthesis in E. coil.

FIG. 3. Reaction steps in the conversion of 3-deoxy-arabino-heptulonate7-phosphate (DHAP) to muconic acid.

FIG. 4. Chromatograph showing standards used for HPLC analysis of totalmuconic acid and biochemical intermediates.

FIG. 5. Chromatograph showing standards used for HPLC analysis ofmuconic acid isomers.

FIG. 6. Titer for the production of DHS in the E. coli strain MYR34transformed with plasmids pCP32AMP, pCP14 and pCP54. MYR34 strain of E.coli has a deletion in aroE gene. The plasmid pCP32AMP expresses thearoG gene coding for DAHP synthase. The plasmid pCP14 expresses the aroBgene coding for DHQ synthase. The plasmid pCP54 expresses both the aroBand aroG genes.

FIG. 7. Titer for the production of DHS in the E. coil strains MYR34 andMYR170 transformed with plasmids pCP32AMP and pCP54. The MYR34 strain ofE. coli has a deletion of the aroE gene. The MYR170 strain has adeletion of the aroE gene and a second copy of the aroB gene under thecontrol of P₁₅ promoter integrated at the ack locus of the hostchromosomal DNA. The plasmid pCP32AMP expresses the aroG gene coding forDAHP synthase. The plasmid pCP54 expresses both aroB and aroG genes.

FIG. 8. Titer for the production of cis, cis-muconic acid in the E. colistrains MYR34 and MYR170 transformed with plasmid pMG37 alone or withboth pMG37 and pCP32AMP plasmids. The MYR34 strain of E. coil has adeletion of the aroE gene. The MYR170 strain has a deletion of the aroEgene and a second copy of the aroB gene under the control of P₁₅promoter integrated at the ack locus of the host chromosomal DNA. Theplasmid pCP32AMP expresses the aroG gene coding for DAHP synthase. Theplasmid pMG37 expresses the aroZ, aroY, and catAX, genes coding forproteins functional in the muconic acid pathway.

FIG. 9. Titer for the production of DHS in MYR170 strain of E. colitransformed with a plasmid expressing aroG gene alone (pCP32AMP) or aroGand tktA genes simultaneously (pCP50). The MYR170 strain has a deletionof the aroE gene and a second copy of the aroB gene under the control ofthe P₁₅ promoter integrated at the ack locus of the host chromosomalDNA.

FIG. 10. DHS yield from MYR34 and MYR170 stains of E. coli transformedwith plasmids pCP32AMP and pCP50. DHS yield is calculated as grams ofDHS produced per gram of glucose consumed. The plasmid pCP32AMPexpresses the aroG gene while pCP50 expresses aroG and tktA. Thebacterial strain MYR34 has a deletion in the aroE gene. The MYR170strain of E. coil is derived from MYR34 and has an additional aroB geneintegrated at the ack locus on the chromosomal DNA.

FIG. 11. DHS titer from MYR170 and MYR261 stains of E. coli transformedwith plasmids pCP32AMP and pCP50. The plasmid pCP32AMP expresses thearoG gene while pCP50 expresses the aroB and tktA genes. The MYR170strain has a deletion of the aroE gene and a second copy of the aroBgene under the control of P₁₅ promoter integrated at the ack locus ofthe host chromosomal DNA. MYR261 strain of E. coli is derived from theMYR170 strain of E. coli. MYR261 strain of E. coli has a second copy ofthe tktA gene with its native promoter integrated at the poxB locus ofthe chromosomal DNA.

FIG. 12. Muconic acid and acetic acid production in the E. coli strainsMYR170, MYR261 and MYR305 transformed with the plasmid pCP32AMPexpressing aroG coding for DAHP synthase in the shikimic acidbiosynthetic pathway and plasmid pMG37 expressing aroZ, aroY and catAXgenes coding for proteins functional in the muconic acid pathway. MYR170strain has a deletion in the aroE gene and an additional copy of aroBgene under the control of the P₁₅ promoter inserted at ack locus in thehost chromosomal DNA. MYR261 and MYR305 are derivatives of MYR170strain. MYR261 has an additional copy of tktA gene integrated at poxBlocus on the host chromosomal DNA while MYR305 has a deletion in thepoxB locus on the host chromosomal DNA.

FIG. 13. Conversion of endogenous DHS produced by E. coli strain MYR34into muconic acid. Strain MYR34 of E. coli has a deletion in the aroEgene coding of shikimate dehydrogenase. As a result there is anaccumulation of DHS. When strain MYR34 is transformed with a plasmidexpressing aroZ, aroY and catAX, genes coding for proteins functional inmuconic acid pathway, there is conversion of DHS into muconic acid.However, no conversion of DHS into muconic acid occurs when MYR34 strainis transformed with the empty plasmid vector (pCL1921) without anyexogenous genes.

FIG. 14. Comparison of aroZ analogs for their ability to divert DHS intothe muconic acid pathway. Three different aroZ analogs, namely quiC fromAcinetobacter sp. ADP1, asbF from Bacillus thuringiensis, and qa-4 fromNeurospora crassa were cloned under the P₂₆ promoter in a low-copyplasmid which also expressed catAX and aroY genes from the P₁₅ andlambda P_(R) promoters respectively. These three different plasmidconstructs were expressed in MYR34 through transformation and the amountof muconic acid produced was measured.

FIG. 15. Single copies of catAX, aroY and quiC were chromosomallyintegrated into MYR170 strain of E. coil (ΔaroE, Δack::P₁₅-aroB)resulting in MYR352 (SEQ ID No. 41). MYR170 was also transformed withlow copy plasmid pMG37 carrying all genes necessary for the operation ofmuconic acid pathway leading to the MYR219 strain. Both MYR352 andMYR219 were transformed with YEp24 (medium-copy empty vector) orpCP32AMP (medium-copy aroG expressing plasmid) or pCP50 (medium-copyaroG and tktA expressing plasmid) and the amount of PCA, catechol andmuconic acid produced were quantified using HPLC method.

FIG. 16. Removal of catechol accumulation in MYR352 by means ofincreasing the expression of catAX. MYR352 was transformed with aplasmid expressing aroY alone (pMG27) or a plasmid expressing quiC alone(pMG39) or a plasmid expressing all three muconic acid pathway genesnamely catAX, aroY and quiC (pMG37) or a plasmid expressing only twogenes in the muconic acid pathway namely catAX and aroY (pMG33). Overexpression of catAX alone was sufficient to prevent accumulation ofcatechol.

FIG. 17. Growth of strains using different systems for importingglucose. Deletion of ptsHI and galP (MYR31) leads to lack of growth inminimal glucose medium, while installation of glf and glk genes (MYR217)brings back growth. Control strain MYR34 is ΔaroE, but otherwise wildtype. The three aromatic amino acids and three aromatic vitamins wereadded to the medium to allow growth of the auxotrophic strains.

FIG. 18. DHS production in MYR34 and MYR217 strains of E. coli. Whentransformed with plasmids that lead to production of DHS, MYR217, whichutilizes glf-glk for glucose import, produced a higher titer of DHS thantransformants of MYR34, which utilizes the phosphotransferase system(PTS).

FIG. 19. Production of muconic acid by the MYR428 strain of E. coli in a7 Liter fermentor. The MYR261 strain of E. coli with a genotype of ΔaroEΔackA::P₁₅-aroB ΔpoxB::tktA was transformed with the plasmids pCP32AMPand pMG37 to generate the MYR428 strain of E. coli.

FIG. 20. Muconic acid and PCA production in the E. coli strain MYR993genetically engineered to produce muconic acid and two MYR993derivatives MYR993ΔubiX and MYR993ΔubiD. The MYR993AubiX E. coil strainwas derived from MYR993 strain by means of replacing the coding regionfor ubiX gene with a cassette coding for kanamycin resistance. TheMYR993ΔubiD E. coli strain was similarly derived from MYR933 strain bymeans of replacing the coding region for ubiD gene with a cassettecoding for kanamycin resistance.

FIG. 21. Measurement of relative activity of various homologs of UbiX.Measurement of activity of UbiX homologs was carried out from thedecarboxylation of PCA as measured by a decrease in the absorbance at290 nm (A290). Five different UbiX homologs namely KpdB, UbiX, Elw, Kokand Lpl were use in this study.

FIG. 22. Muconic acid and PCA production in E. coli strains having lowor high level of kpdB gene expression. E. coli strain MYR1305 with noexogenous kpdB gene was used as the parent strain and was transformedwith a low copy plasmid having the kpdB gene expressed either from theP26 promoter or the E. coli pgi promoter. The gene expression from theP26 promoter is expected to be at a relatively low level while the geneexpression from the pgi promoter is expected to be at a relatively highlevel.

FIG. 23. Muconic acid production in the E. coli strain MYR1674 and itsderivative MYR1772. MYR1772 was derived from MYR1674 by replacing thecoding region and promoter of ppc with the P_(R)-pyc gene. P_(R) is anabbreviation for the strong rightward promoter from the coliphagelambda.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All the patents, patent applications, publications, sequences, and otherpublished materials are incorporated herein are incorporated byreference.

As used in this patent application, the phrase “for example” or “suchas” is meant to indicate that there arc more than one method, approach,solution, or composition of matter for the subject at hand, and theexample given is not meant to be limiting to that example.

The term “heterologous” refers to a gene or protein that is notnaturally or natively found in an organism, but which can be introducedinto an organism by genetic engineering, such as by transformation,mating, or transduction. A heterologous gene can be integrated(inserted) into a chromosome or contained on a plasmid. The term“exogenous” refers to a gene or protein that has been introduced into,or altered, in an organism for the purpose of increasing, decreasing, oreliminating an activity, by genetic engineering, such as bytransformation, mating, transduction, or mutagenesis. An exogenous geneor protein can be heterologous, or it can be a gene or protein that isnative to the host organism, but altered by one or more methods, forexample, mutation, deletion, change of promoter, change of terminator,duplication, or insertion of one or more additional copies in thechromosome or in a plasmid. Thus, for example, if a second copy of thearoB gene is inserted at a site in the chromosome that is distinct fromthe native site, the second copy would be exogenous.

The term “microorganism” as used in this present invention includesbacteria, archaea, yeast, algae and filamentous fungi that can be usedfor the commercial production of cis, cis-muconic acid through afermentation process. The term “genetically engineered microorganism”refers to microorganisms that are not present in the Nature butgenerated using one or other genetic modifications as described in thispatent application.

For nomenclature, a gene or coding region is usually named with lowercase letters in italics, for example “aroZ”, while the enzyme or proteinencoded by a gene can be named with the same letters, but with the firstletter in upper case and without italics, for example “AroZ”. The enzymeor protein can also be referred to by a more descriptive name, forexample, AroZ can also be referred to as 3-dehydroshikimate dehydratase.A gene or coding region that encodes one example of an enzyme thatpossess a particular catalytic activity can have several different namesbecause of historically different origins, or because the gene comesfrom different species. For example the gene that encodes3-dehydroshikimate dehydratase from Bacillus anthracis can be named asbFinstead of aroZ, the same gene from Aspergillus nidulans can be namedqutC, the same gene from Neurospora crasser can be named qa-4, and thesame gene from Acinetobacter baylyi can be named quiC.

A “plasmid” means a circular or linear DNA molecule that issubstantially smaller than a chromosome, separate from the chromosome orchromosomes of a microorganism, and that replicates separately from thechromosome or chromosomes. A “plasmid” can be present in about one copyper cell or in more than one copy per cell. Maintenance of a plasmidwithin a microbial cell in general requires an antibiotic selection, butcomplementation of an auxotrophy can also be used.

The term “chromosome” or “chromosomal DNA” as used in this invention inthe context of a bacterial cell is a circular DNA molecule that issubstantially larger than a plasmid and does not require any antibioticsselection.

The term an “expression cassette” or a “cassette” means a DNA sequencethat can be part of a chromosome or plasmid that contains at least apromoter and a gene or region that codes for an enzyme or other protein,such that the coding region is expressed by the promoter, and the enzymeor protein is produced by a host cell that contains the DNA sequence. An“expression cassette” can be at least partly synthetic, or constructedby genetic engineering methods, so that the coding region is expressedfrom a promoter that is not naturally associated with the coding region.Optionally, the “expression cassette” can contain a transcriptionterminator that may or may not be a terminator that is naturallyassociated with the coding region. An “expression cassette” can havecoding regions for more than one protein, in which case it can be calledan operon, or a synthetic operon.

The term “overexpression” of a gene or coding region means causing theenzyme or protein encoded by that gene or coding region to be producedin a host microorganism at a level that is higher than the level foundin the wild type version of the host microorganism under the same orsimilar growth conditions. This can be accomplished by, for example, oneor more of the following methods: 1) installing a stronger promoter, 2)installing a stronger ribosome binding site, such as a DNA sequence of5′-AGGAGG, situated about four to ten bases upstream of the translationstart codon, 3) installing a terminator or a stronger terminator, 4)improving the choice of codons at one or more sites in the codingregion, 5) improving the mRNA stability, and 6) increasing the copynumber of the gene, either by introducing multiple copies in thechromosome or placing the cassette on a multicopy plasmid. An enzyme orprotein produced from a gene that is overexpressed is said to be“overproduced”. A gene that is being “overexpressed” or a protein thatis being “overproduced” can be one that is native to a hostmicroorganism, or it can be one that has been transplanted by geneticengineering methods from a different organism into a host microorganism,in which case the enzyme or protein and the gene or coding region thatencodes the enzyme or protein is called “foreign” or “heterologous”.Foreign or heterologous genes and proteins are by definitionoverexpressed and overproduced, since they are not present in theunengineered host organism.

The term a “homolog” of a first gene, DNA sequence, or protein is asecond gene, DNA sequence, or protein that performs a similar biologicalfunction to that of said first gene, DNA sequence or protein, and thathas at least 25% sequence identity (when comparing protein sequences orcomparing the protein sequence derived from gene sequences) with saidfirst gene or protein, as determined by the BLAST computer program forsequence comparison (Altschul et al., 1990; Altschul et al., 1997),using default parameters and allowing for deletions and insertions. Anexample of a homolog of the E. coli aroG gene would be the aroG genefrom Salmonella typhimurium.

Two enzymes or proteins that are very distantly related by homology cancarry out the same biochemical function but be only relatively weaklyhomologous to each other. For example, the FurnA fumarase from E. coliK-12 (GenBank NP_416129) is about 26.9% homologous to a fumarase fromClostridium botulinum (GenBank GAE03909.1) over their region of overlap,and about 25.1% homologous to a fumarase beta subunit from Pyrococcussp. ST04 (GenBank AKF23146.1) over their region of overlap. As anotherexample, a Klebsiella AroZ and a Neurospora crassa Qa-4 enzyme that bothfunction to convert DHS to PCA are 29.3% identical. Therefore, since forthe genetic engineering of a metabolic pathway, the important feature ofa heterologous enzyme or protein is the function or reaction carried outby that enzyme or protein, not the source organism or the precise aminoacid sequence, we define “homologous” enzymes or proteins or “homologs”to comprise any pair of enzymes or proteins that are 25% or higher inthe identity of their amino acid sequences, allowing for gaps, as shownfor example by using the default parameters for alignment using theLaserGene 12 (DNAStar, Madison, Wis.) MegAlign program with theLipman-Pearson method (Ktuple=2, Gap penalty=4, and Gap lengthpenalty=12).

The term an “analog” of a first gene, DNA sequence, or protein is asecond gene, DNA sequence, or protein that performs a similar biologicalfunction to that of said first gene, DNA sequence, or protein, but wherethere is less than 25% sequence identity (when comparing proteinsequences or comparing the protein sequence derived from gene sequences)with said first gene, DNA sequence or protein, as determined by theBLAST computer program for sequence comparison (Altschul et al., 1990;Altschul et al., 1997), and allowing for deletions and insertions. Anexample of an analog of the Klebsiella pneumoniae AroZ protein would bethe QutC protein from Aspergillus nidulans, since both proteins areenzymes that catalyze the 3-dehydroshikimate dehydratase reaction, butthere is no significant sequence homology between the two enzymes ortheir respective genes. A scientist knowledgeable in the art will knowthat many enzymes and proteins that have a particular biologicalfunction, for example DAHP synthase or 3-dehydroshikimate dehydratase,can be found in many different organisms, either as homologs or analogs,and since members of such families of enzymes or proteins share the samefunction, although they may be slightly or substantially different instructure, different members of the same family can in many cases beused to perform the same biological function using current methods ofgenetic engineering. Thus, for example, the AroZ enzyme and the QutCenzyme catalyze the same reaction, DHS dehydratase, so either one willresult in production of cis, cis-muconic acid in the proper context, andthe choice of which one to use ultimately can be made by choosing theone that leads to a higher titer of cis, cis-muconic acid under similarfermentation conditions.

The terms a “non-aromatic carbon source” or a “non-aromatic compound”means a carbon-containing compound that can be used to feed amicroorganism of the invention as a source of carbon and/or energy, inwhich the compound does not contain a six-membered ring related tobenzene. Examples of non-aromatic carbon sources include glucose,xylose, lactose, glycerol, acetate, arabinose, galactose, mannose,maltose, or sucrose. An “aromatic compound” is a compound that containsone or more six-membered rings related to benzene. An example of anaromatic compound is catechol, or 1,2-dihydroxy benzene. A microorganismselected for producing muconic acid using glucose as a source ofnon-aromatic carbon source can further be engineered to use other typesof non-aromatic carbon sources such as glycerol, sucrose and xyloseusing the genetic engineering techniques as provided in the patentdocuments U.S. Pat. No. 8,871,489 and US Patent Application PublicationNos. US2013/0337519A1 and US2014/0234923A.

The term a “strong constitutive promoter” means a DNA sequence thattypically lies upstream (to the 5′ side of a gene when depicted in theconventional 5′ to 3′ orientation), of a DNA sequence or a gene that istranscribed by an RNA polymerase, and that causes said DNA sequence orgene to be expressed by transcription by an RNA polymerase at a levelthat is easily detected directly or indirectly by any appropriate assayprocedure. Examples of appropriate assay procedures include 1)quantitative reverse transcriptase plus PCR, 2) enzyme assay of anencoded enzyme, 3) Coomassie Blue-stained protein gel, or 4) measurableproduction of a metabolite that is produced indirectly as a result ofsaid transcription, and such measurable transcription occurringregardless of the presence or absence of a protein that specificallyregulates level of transcription, a metabolite, or inducer chemical. Anexample of a promoter that is nota “strong constitutive promoter” is theP_(lac) promoter of E. coli, since it is repressed by a repressor in theabsence of lactose or the inducer IPTG. By using well known methods inthe art, a “strong constitutive promoter” can be used to replace anative promoter (a promoter that is otherwise naturally existingupstream from a DNA sequence or gene), resulting in an expressioncassette that can be placed either in a plasmid or chromosome and thatprovides a level of expression of a desired DNA sequence or gene at alevel that is higher than the level from the native promoter. A strongconstitutive promoter can be specific for a species or genus, but oftena strong constitutive promoter from a bacterium can function well in adistantly related bacterium. For example, a promoter from Bacillussubtilis or a phage that normally grows on B. subtilis can function wellin E. coli. A “strong constitutive promoter” is substantially differentfrom inducible promoters, such as P_(tac), which have been used in theprior art production of cis, cis-muconic acid and typically require anexpensive chemical or other environmental change for the desired levelof function (Niu et al., 2002), Examples of strong constitutivepromoters are P₁₅, P₂₆, from Bacillus subtilis phage SP01, and coliphage lambda P_(R).

A “mutation” is any change in a DNA sequence that makes it differentfrom a related wild type sequence. A “mutation” can comprise a singlebase change, a deletion, an insertion, a replacement, a frameshift, aninversion, a duplication, or any other type of change in a DNA sequence.Usually a “mutation” refers to a change that has a negative effect onfunction or reduce the activity of a gene or gene product, however,herein, the term “mutation” can also refer to a change that increasesthe activity of a gene or gene product. For example, a feedbackresistant mutation in the aroG gene increases the activity of AroG inthe presence of an inhibitor such as phenylalanine. Replacement of apromoter with a different, stronger promoter, also result in a mutationthat can increase the activity of a gene or gene product. A “nullmutation” is a mutation, such as a deletion of most or all of a gene, isa mutation that effectively eliminates the function of a gene. A“mutant” is a strain or isolate that comprises one or more mutations.

The biological production of cis, cis-muconic acid (herein referred toas simply “muconic acid”) is based on the redirection of carbon from thearomatic amino acid pathway. Native production of aromatic amino acidsand vitamins requires the metabolites erythrose-4-phosphate (E4P) andphosphoenolpyruvate (PEP). The first committed step in aromatic aminoacid synthesis is catalyzed by the enzyme 3-deoxy-arabino-heptulonate7-phosphate (DAHP) synthase. In E. coli, this step can be performed bythree different isozymes, AroG, AroF, or AroH. Each of these enzymes isregulated at the transcriptional level by a repressor protein TyrR, aswell as at the protein level by inhibition from terminal products of thepathway, phenylalanine, tyrosine, and tryptophan, respectively. Theproduction of muconic acid proceeds from an intermediate in the aromaticamino acid pathway, dehydroshikimic acid (DHS) and requires theexpression of three heterologous enzymes, dehydroshikimate dehydratase(AroZ), 3,4-dihydrobenzoate decarboxylase (AroY), and catechol1,2-dioxygenase (CatA). This pathway is shown in FIGS. 1 and 2.

A “muconic pathway” or “muconic acid pathway” refers to a biochemicalpathway from DHS to PCA to catechol to cis, cis-muconic acid, and a“muconic pathway gene” is a gene that encodes an enzymes that catalyzesa step in a muconic pathway, or encodes an auxiliary function thatserves to enhance the activity of one of said enzymes, for example,aroZ, aroY, catA, catX, and qutC (FIG. 3). DHS is an abbreviation for3-dehydroshikimate, and PCA is an abbreviation for protocatechuic acid.A “muconic plasmid” is a plasmid that contains one or more muconicpathway genes.

The genetic manipulations used in the present inventions are centeredaround the common pathway for aromatic amino acid and aromatic vitamin(or vitamin-like) biosynthesis present in many microbial cells as shownin FIG. 1. The common pathway for aromatic amino acid biosynthesis asdepicted in FIG. 1 can be referred to as the “shikimic acid” or“shikimate” pathway, the “chorismic acid” or “chorismate” pathway, orthe “central aromatic” or “central aromatic biosynthetic” pathway.

There is a substantial volume of published work on genetic engineeringof microorganisms for the production of the aromatic amino acids,phenylalanine, tyrosine, and tryptophan (U.S. Pat. Nos. 4,681,852,4,753,883, 6,180,373, European Patent Application 86300748.0). Theapproaches for the production of aromatic amino acids include usingvarious combinations of feedback resistant enzymes (AroF, AroG, PheA,TyrA), deregulation of repression of transcription (tyrR⁻), increasingpromoter strength (P_(tac), P_(lac)) and increasing the copy number ofone or more genes (tktA). Many specific combinations of the abovementioned genetic modifications can be followed to obtain a biocatalystsuitable for muconic acid production.

According to the disclosure in the International Patent ApplicationPublication No. WO2013/116244, incorporated herein in its entirety byreference, the genetically engineered microorganisms do not need tocontain any exogenous plasmids in order to produce muconic acid,although they have certain exogenous or heterologous genes necessary toachieve the desired phenotype. In the preferred embodiment of thepresent invention, the exogenous genes introduced into themicroorganisms are stably integrated into the chromosomal DNA. As aresult of this chromosomal DNA integration of the exogenous genes, theneed for the use of antibiotics or other selective methods to maintainthe plasmids carrying exogenous DNA is totally eliminated. In addition,strong promoters that do not require chemical inducers are used toexpress genes necessary for the operation of the pathway from carbonsource, such as glucose, to cis, cis-muconic acid.

When an exogenous coding sequence is integrated into the chromosomalDNA, it is integrated at a locus, the deletion of which has beenreported not to cause any adverse effects. For example, the codingregion at the physical location 0039 in E. coli bacterium, also known asydeM, is annotated as a lipoprotein and has been demonstrated to have noadverse effects upon deletion. Similarly, the coding region at thephysical location 2160 in E. coli bacterium, also known as nlpA, isannotated as a radical SAM domain protein and has been demonstrated tohave no adverse effects upon deletion. In the present invention, a copyof P_(R)-catAX was inserted at the physical location 0039 in E. colibacterium and a copy of P_(R)-aroG^(FBR) was inserted at the physicallocation 2160 in E. coil bacterium. In other examples described herein,an insertion is made in gene that is advantageous to knockout ordeleted, such as a gene encoding for an unwanted function, for example aptsI gene or a tyrR gene.

The aromatic amino acid biosynthetic pathway is well known for manymicroorganisms, especially for E. coil (Neidhart and Curtiss 1996). In awild type cell, the pathway is tightly regulated by both feedbackinhibition and repression of transcription. The first committed step iscatalyzed by deoxy-arabino-heptulosonate 7-phosphate (DAHP) synthase, ofwhich there are three isozymes encoded by aroF, aroG, and aroH. Thethree isozymes, AroF, AroG, and AroH, are feedback inhibited by theproducts of aromatic amino biosynthetic pathway namely by tyrosine,phenylalanine, and tryptophan. Feedback resistant mutants of AroF, AroG,and AroH are well known (Flu et al. 2003; Lutke-Eversloh andStephanopoulos 2007). One aspect of the present invention involves useof feedback resistant alleles of aroF, aroG, and aroH genes in order toexpress AroF, AroG and AroH enzyme proteins that are resistant tofeedback inhibition by the products of aromatic amino acid biosyntheticpathway. The AroF, AroG and AroH enzyme proteins that are resistant tofeedback inhibition are referred as AroF^(FBR), AroG^(FBR), andAroH^(FBR).

Transcription of several of the operons involved in the aromatic pathwayis regulated by either the repressor encoded by the tyrR gene or therepressor encoded by the trpR gene, or both (Neidhardt and Curtiss1996). Of particular importance is the negative regulation oftranscription of aroG and aroF by the TyrR protein when it is bound withone or more of the aromatic amino acids. One aspect of the presentinvention involves the removal of negative regulation by tyrR or trpRgenes by means of eliminating these genes from the chromosome of thehost bacterial strains.

The present invention teaches certain combinations of genetic elementsin the biocatalysts suitable for muconic acid production, for example,but not limited to, various combinations of an overproduced feedbackresistant AroG, an overproduced feedback resistant AroF, anoverexpressed tktA, an overexpressed talA, chromosomally integratedcassettes for expressing an aroZ, aroY, and a catAX (or analogs orhomologs thereof) from strong constitutive promoters, and a leaky aroEallele, which we define as a gene that encodes an AroE enzyme thatconfers prototrophy for the aromatic amino acids and vitamins, butwithout leading to significant secretion of unwanted aromatic compounds.

All specific examples of strain constructions disclosed herein are basedon wild type Escherichia coli C strain (ATCC 8739), or Escherichia coliW strain (ATCC 9637). However, it should be realized at this point thatthe expression cassettes or appropriate analogs and homologs of thegenetic elements disclosed herein can be assembled in any other suitablemicroorganism, such as any other suitable E. coil strains and otherspecies of bacteria, archaea, yeast, algae, and filamentous fungi thatcan be used for the commercial production of muconic acid through afermentative process.

In E. coli, the aromatic amino acid biosynthesis pathway from glucosestarts with the non-oxidative branch of the pentose phosphate pathway(PPP). Four key enzymes in the non-oxidative pentose phosphate pathwayare transketolase, transaldolase, ribulose-5-phosphate epimerase andribulose-5-phosphate isomerase. These enzymes catalyze the reactionsthat lead to the formation of erythrose 4-phosphate (E4P) from hexose orpentose sugars. To increase the availability of E4P in E. coil, the tktAgene encoding transketolase can be overexpressed (Niu et al., 2002).Similarly, the overexpression of the transaldolase gene is also expectedto increase the availability of E4P in some circumstances (Bongaerts etal., 2001). In yet another aspect of the present invention, theexpression of both the transketolase and transaldolase genes areenhanced through genetic manipulations leading to an increase in theactivity of transketolase and transaldolase enzymes. In yet anotheraspect of the invention, flux through the non-oxidative branch of thePPP is increased by overproducing ribulose-5-phosphate epimerase andribulose-5-phosphate isomerase.

The first committed step and most tightly regulated reaction in thecommon aromatic amino acid pathway is the condensation ofphosphoenolpyruvate (PEP) and E4P to produce deoxyarabino-heptulosonate7-phosphate (DAHP) by DAHP synthase (encoded by aroG, aroF, and aroH),D-glucose consumed by E. coli is brought into aromatic biosynthesispartly through the PPP, and partly through glycolysis. The flow ofglucose into the aromatic pathway is greatly increased whentransketalose (tktA) and an isozyme of DAHP synthase (aroG) areamplified through transformation with a plasmid that increases theirexpression by increasing their copy number (Niu et al., 2002). In apreferred aspect of the present invention, the exogenous aroG and tktAgenes are integrated into the chromosomal DNA for the purpose ofamplification of activities transketolase and DAHP synthase enzymes.

In another embodiment of the present invention, the flux through PEPwithin the microbial cell is improved by increasing the PEP availablefor the synthesis of DAHP by reducing the flux of PEP to other pathways.Many genera of bacterial cells consume PEP in the transport of glucoseacross the cell membrane using a phosphotransferase system (PTS) inwhich one PEP molecule is consumed for every molecule of glucosetransported across the bacterial outer membrane. By replacing orcomplementing the PEP-dependent PTS with a non-PEP dependent (PEPindependent) glucose uptake mechanisms, it is possible to increase thepool size of the PEP available for the aromatic amino acid biosyntheticpathway within the microbial cell. For example, the PTS system for sugaruptake can be replaced or complemented by a GalP-based sugar uptakesystem or the sugar transporter system based on Glf/Glk proteins(Chandran et al., 2003; Yi et al., 2003). In a preferred aspect of thepresent invention besides deleting the PTS system for sugar uptake forthe purpose of conserving PEP pool within the microbial cell, the GalPbased sugar uptake system is also inactivated for the purpose ofconserving ATP within the microbial cell. In a microbial cell which isdefective in the functioning of both PTS system and a Gal-P based sugaruptake system (ΔPTS/ΔgalP), the sugar uptake can be accomplished bymeans of introducing an exogenous gene coding for Glf (glucosefacilitated diffusion protein), or exogenous genes encoding both Glf andGlk (glucokinase) proteins. As used in the present invention, the termfunctional glucose-facilitated diffusion protein refers to any Glfprotein as well as any other protein which is functionally equivalent toGlf and functions to transport sugars into the microbial cells byfacilitated diffusion. In one aspect of the present invention, the genecoding for the glucose facilitator protein Glf is introduced into themicrobial cell which is ΔPTS/ΔgalP and the glucose transported into themicrobial cell is phosphorylated by endogenous glucose kinase. Inanother aspect of the present invention the genes coding for both Glfand Glk proteins are introduced into a microbial cell which isΔPTS/ΔgalP. In a preferred aspect of the present invention, theexogenous glf and glk genes introduced into the microbial cell areintegrated into the host chromosomal DNA.

In another embodiment of the present invention, when the carbon sourcefor growth and energy requires gluconeogenesis (for example if thecarbon source is acetate or succinate), the PEP pool can be increased byincreasing the activity of carboxylating enzymes already present withinthe cell, for example PEP carboxykinase, which is encoded by pck in E.coli, or by introducing an exogenous carboxylating enzyme. In apreferred embodiment, the introduced exogenous gene coding for acarboxylating enzyme is stably integrated into the host chromosome.Genes coding for the carboxylating enzyme can be derived from a varietyof microbial species. The genes coding for the carboxylating enzymes canfurther be subjected to genetic manipulations so that the expression ofthe carboxylating enzyme within the biocatalyst for cis, cis-muconicacid production is significantly enhanced.

PEP is one of two major metabolites for the aromatic pathway andreduction or elimination of Ppc activity preserves PEP for the aromaticpathway and muconic acid production. Ppc catalyzes the anapleroticreaction forming oxaloacetate, an intermediate in the TCA cycle. Ppcactivity is essential for wild type E. coli and some other organisms inminimal media, but it is absent in others. Some organisms, such as yeastthat lack Ppc, utilize Pyc to replenish oxaloacetate. Lowered or absentPpc activity can be complemented with Pyc activity, by providing analternate route to oxaloacetate from pyruvate instead of PEP, whichresults in increased availability of PEP for muconic acid production aswell as for production of other compounds, such as aromatic compounds,that require PEP as an intermediate. In at least one example disclosedherein, substituting Pyc for Ppc can reduce flux from PEP to OAA, whichin turn conserves PEP for the central aromatic pathway.

In yet another embodiment of the present invention, the PEP pool insidethe microbial cell is increased by decreasing or eliminating theactivity of pyruvate kinase enzymes such as PykA and PykF which use PEPas a substrate.

From DAHP, the aromatic amino acid pathway proceeds via a number ofintermediates to chorismate (CHA), a branch point for the biosynthesisof three aromatic amino acids namely L-Tyrosine (L-Tyr), L-Phenylalanine(L-Phe), and L-Tryptophan (L-Trp).

In the initial stages of the common aromatic amino acid pathway,3-dehydroquinate (DHQ) synthase (AroB) removes the phosphate group fromDAHP leading to the formation of DHQ. The enzyme DHQ dehydratase (AroD)removes a water molecule from DHQ leading to the formation of3-dehydroshikimate (DHS) which is subsequently reduced to shikimate(SHK) by shikimate dehydrogenase (AroE). Shikimate kinase I/II (AroK,AroL) phosphorylates shikimate to shikimate 3-phosphate (S3P). There isa condensation of S3P with PEP leading to the formation of 5enolpymvoylshikimate 3-phosphate (EPSP). The formation of EPSP ismediated by EPSP synthase (AroA). A phosphate group from EPSP is removedby chorismate synthase (AroC) leading to the formation of chorismate(CHA).

As shown in the FIG. 2, the aromatic amino acid pathway can be blockedat the level of conversion of 3-dehydroshikimate (DHS) to shikimate(SHK) due to a mutation in an aroE gene leading to the accumulation ofDHS (Niu et al., 2002). Introduction of an exogenous aroZ gene functionsto convert DHS into protocatechuate (PCA). PCA is subsequently convertedinto catechol through a decarboxylation reaction mediated by an AroYenzyme. Catechol is ultimately converted into cis-cis-muconic acid(ccMuA) through the action of a catA gene product. ccMuA can be actedupon by maleyl acetoacetate isomerase to yield trans-trans muconic acid(ttMuA). The biosynthetic pathway from DHS to ccMuA and/or ttMuA isreferred to as a muconic acid pathway. The three different genesresponsible for the conversion of DHS to ccMuA can be obtained fromvarious microbial species and introduced into a microorganism selectedfor muconic acid production such as Escherichia coli. In a preferredembodiment of the present invention, the exogenous genes coding for theproteins involved in muconic acid pathway are integrated into hostchromosomal DNA.

In redirecting the aromatic amino acid pathway to the production of cis,cis-muconic acid, the mutation of the aroE gene is critical. The aroEgene can be completely inactivated leading to a total block in thebiosynthesis of aromatic amino acids as was done with theWN1/pWN2.248strain of E. coil described for the muconic acid production(Niu et al., 2002). An important drawback with the WN1/pWN2.248 E. coilstrain and related strains is that due to the complete inactivation ofthe aroE gene, this strain has become auxotrophic for the aromatic acidssuch as phenylalanine, tyrosine and tryptophan, and aromatic vitamins orvitamin-like compounds mentioned above. As a result, this strain duringits growth for the production of cis, cis-muconic acid requires theexogenous addition of these compounds (or a common intermediate such asshikimate), thereby adding substantially to the cost of commercialproduction of cis, cis-muconic acid using such a strain. A novelapproach to overcome this dependency on an exogenous source of aromaticamino acids is to use a strain with a leaky mutation in aroE. The leakyaroE mutant would allow a limited flow of carbon to shikimic acid whileaccumulating significant amounts of DHS which is then available for theconversion into PCA by the action of an AroZ enzyme. Thus the use of aleaky mutant form of aroE would eliminate the dependence on exogenousaromatic amino acids, while still diverting the flow of carbon to cis,cis muconic acid.

The genes coding for the synthesis of AroZ, AroY and CatA proteinsessential for the conversion of DHS into cis, cis-muconic acid can bederived from any one of many microbial species. In one embodiment, theseexogenous genes are integrated into the host chromosome of thebiocatalyst being developed. In a preferred embodiment, the expressionof these exogenous genes within the biocatalyst is driven by aconstitutive promoter without the need for any inducers.

The enzyme 3-dehydroshikimate dehydratase (AroZ; EC 4.2.1.118) isrequired for biosynthesis of the intermediate protocatechuate. In thisspecification, “AroZ” shall refer to any enzyme that catalyzes the3-dehydroshikimate dehydratase reaction. In the prior art, this enzymeis expressed from the aroZ gene of Klebsiella pneumoniae strain A170-40(ATCC25597) (Niu et al., 2002; Draths and Frost, 1995). However, thespecific activity of AroZ varies widely among organisms, from 0.1 to 261micromoles/min/mg (Wheeler et al, 1996; Fox et al, 2008; Pfleger et al,2008), so a significant improvement can be had by expressing an aroZgene also known as asbF (Fox et al, 2008; Pfleger et al, 2008), qutC(Wheeler et al, 1996), qa-4 (Rutledge, 1984), and quiC, from an organismthat has a higher specific activity than K pneumoniae, for exampleAcinteobacter baylyi, Aspergillus nidulans (Wheeler et al, 1996), nowalso known as Emericella nidulans, or Neurospora crassa (Rutledge, 1984;Stroman et al, 1978), or Podospora anserina, also known as Podosporapauciseta (Hansen et al, 2009).

As one particular example, the coding sequence for the qa-4 gene from N.crassa that encodes 3-dehydroshikimate dehydratase can be obtained byany of several well-known methods, for example whole gene DNA synthesis,cDNA cloning, or by a combination of genomic DNA cloning and PCR orsynthetic DNA linker synthesis. Since there are no introns in the qa-4gene, the coding region can be obtained by PCR from genomic DNA(Rutledge, 1984). The protein sequence of the qa-4 enzyme (SEQ ID No. 4)and the DNA sequence of the native gene (SEQ ID No. 5) are known.

Alternatively, an expression cassette can be constructed for the3-dehydroshikimate dehydratase from A. nidulans. The coding sequence forthe QutC enzyme from A. nidulans can be obtained by any of severalwell-known methods, for example whole gene DNA synthesis, cDNA cloning,or by a combination of genomic DNA cloning and PCR or synthetic DNAlinker synthesis. The protein sequence of QutC (SEQ ID No. 6) and theDNA sequence of the native gene, containing no introns, are known (SEQID No. 7; GenBank accession number M77665.1). An expression cassette canbe obtained by DNA synthesis, or by a combination of genomic cloning andPCR, so that the QutC enzyme can be produced accurately in E. coil. Byexpressing a coding sequence for QutC from a strong, constitutivepromoter in E. coil, sufficient expression can be obtained from one ortwo copies of the gene integrated in the chromosome, obviating the needfor maintaining more than two copies of the expression cassette on amulticopy plasmid as has been disclosed in the prior art (Niu et al.,2002), and which can lead to instability. The method described above canbe used in general to obtain a DNA sequence that codes for a desiredenzyme, and that coding sequence can then be used to construct anexpression cassette designed to function in E. coli or anotherappropriate microbial host organism.

The specific activity of AroZ can also be improved by using the proteinsequence from the prior art (Niu et al., 2002) by constructing animproved expression cassette, for example, in which a stronger promoterand/or ribosome binding site (RBS) has been installed in front of thecoding region, as described in Example 4.

The aroZ gene encoding AroZ (3-dehydroshikimate dehydratase) fromKlebsiella pneumoniae strain A170-40 can be obtained as described in theprior art. The DNA sequence of the gene and surrounding DNA can bedetermined by methods well known in the art. A heterologous gene of theinvention such as aroZ can be built into an expression cassette using anative DNA sequence or it can be synthesized with a codon optimizedsequence for the intended host organism. An aroZ gene can be cloned asdescribed (Draths and Frost, 1995) from any other microbe that containsan active aroZ gene, for example K. pneumoniae strain 342, AcinetobacerSp. ADP1 (Acinetobacter baylyi ADP1), Bacillus thuringiensis, Emericellanidulans, Erwinia amylovora, Pseudomonas putida W619, Neurospora crassa,Aspergillus nidulans and many others.

The enzyme protocatechuate decarboxylase (AroY; EC 4.1.1.63) is requiredfor biosynthesis of the intermediate catechol. In this specification,“AroY” shall refer to any enzyme that catalyzes the protocatechuatedecarboxylase reaction. In the prior art, this enzyme is expressed fromthe aroY gene of Klebsiella pneumoniae strain A170-40 (ATCC25597) on amulticopy plasmid (Niu et al., 2002). However, once again an improvementin the process can be gained by producing enough of the enzyme from oneor two copies of an expression cassette integrated in the chromosome ofthe host organism. This can be accomplished by obtaining an aroY genefrom an organism that naturally produces an AroY enzyme that has higherspecific activity than that of the K. pneumoniae AroY enzyme of theprior art, or by increasing the level of expression of the K. pneumoniaeAroY by constructing an expression cassette that, for example, uses astrong constitutive promoter and/or strong RBS as described above underExample 4. The protein sequence for AroY from K. pneumoniae strainA170-40 is given in SEQ ID No. 8. The corresponding gene, aroY, can becloned as described above (Draths and Frost, 1995), or based on theprotein sequence, it can be synthesized with optimized codons for theintended host organism.

The aroY gene can be obtained from any other microorganism that containsa homolog or analog, for example, K. pneumoniae strain NCTC418(ATCC15380), Klebsiella pneumoniae 342, and Arxula adeninivorans(Sietmann et al, 2010). The DNA sequence of the aroY gene fromKlebsiella pneumoniae 342 and surrounding DNA is given as SEQ ID No. 9.

The enzyme catechol 1,2-dioxygenase (CatA; EC 1.13.11.1) is required forthe last step of cis, cis-muconic acid biosynthesis. In thisspecification, “CatA” shall refer to any enzyme that catalyzes thecatechol 1,2-dioxygenase reaction. In the prior art, this enzyme isexpressed from the catA gene of Acinetobacter calcoaceticus strain ADP1on a multicopy plasmid (Niu et al., 2002). The source strain,Acinetobacter calcoaceticus strain ADP1, apparently has been renamedAcinetobacter Sp. ADP1 and Acinetobacter baylyi ADP1 (Neidle and Omston,1986; Barbe et al, 2004; de Berardinis et al, 2008). In this prior artexample, the catA gene was expressed from a P_(tac) promoter, whichrequires either lactose or IPTG (isopropylthiogalactoside) as aninducer. These compounds are too expensive for use in commercialfermentations, so again, significant improvements in the process areneeded, both to eliminate the need for an expensive inducer and tocreate a more stable strain by integrating the expression cassette inthe chromosome. This can be accomplished by constructing an expressioncassette for the catA gene that uses a strong constitutive promoter,strong RBS, and/or more stable mRNA as described above in the otherExamples.

The DNA sequence of the catA gene and surrounding sequences fromAcinetobacter baylyi ADP1 is given in SEQ ID No. 10. The proteinsequence for CatA from the same strain is given in SEQ ID No. 11. In apreferred embodiment, the expression cassette for catA contains one ortwo additional open reading frames that exist naturally downstream fromcatA, in order to increase the expression level of the catA gene(Schirmer and Hillen, 1998). Many other organisms can be a source for acatA gene, for example Pseudomonas arvilla, Pseudomonas fluorescens(Nakazawa et al, 1967; Kojima et al, 1967), Streptomyces Sp. Strain 2065(Iwagami et al, 2000), Cupriavidus necator 335T, and many others(Perez-Pantoja et al, 2008).

In order to improve the flow of carbon towards cis, cis-muconic acid, itis necessary to block certain other pathways branching out of thearomatic amino acid pathway, besides reducing the flow of carbon fromDHS to shikimate (SHK) by using a leaky aroE mutant. Some bacteria, forexample in the genus Acinetobacter and Pseudomonas, contain a gene namedpobA, which encodes an enzyme, p-hydroxybenzoate hydroxlase, thatconverts DHS into gallic acid. Although a PobA homolog or analog has notbeen found in E. coli, strains of E. coli engineered to produce DHSsecrete measurable amounts of gallic acid (Li and Frost, 1999), so it islikely that such an enzyme does exist in E. coli. In addition, the PCAderived from DHS can be converted into gallic acid by the action ofp-hydroxybenzoate hydroxlase (PobA) enzyme coded by the pobA gene. Thegallic acid thus produced can be subsequently converted to pyrogallol.One way to block the carbon flow to gallic acid and pyrogallol in thebiocatalyst selected for an improved cis, cis-muconic acid is to blockor diminish the activity of p-hydrobenzoate hydroxlase (PobA) proteinthrough genetic manipulations. Similarly, DHQ, the precursor to DHS canalso be acted upon by shikimate dehydrogenase coded by aroE leading tothe production of quinnic acid. In an embodiment of the presentinvention, the leaky AroE mutant enzyme is additionally selected orscreened for its inability or reduced ability to convert DHQ intoquinnic acid.

There are several advantages in producing trans, trans-muconic acid inplace of cis, cis-muconic acid. Trans, trans-muconic acid is preferredover cis, cis-muconic acid in the Diels Alder reaction with ethylene forthe production of terephthalic acid. A biocatalyst with a geneticallymanipulated aromatic pathway produces cis, cis-muconic acid which can beconverted into trans, trans-muconic acid outside the cell using chemicalconversion processes. On the other hand by means of introducing amaleylacetoacetate isomerase or similar isomerase enzyme into thebiocatalyst, it is possible to convert the cis, cis-muconic acid intotrans, trans-muconic acid within the bacterial biocatalyst.

In one embodiment, the present invention provides a genetic approach toenhance the activity of AroY protein involved in the conversion of3,4-dihydroxybenzoic acid (protocatechuic acid or PCA) to catechol. Theactivity of AroY protein has been identified as a major limitation andbottleneck in the biological conversion of glucose to muconic acid(Horwitz et al 2015; Weber et al 2012; Curran et al 2013; Sonoki et al2014). AroY belongs to a class of non-oxidative decarboxylases that arewidespread among many bacteria and utilize a wide-variety of substrates(Lupa et al 2005). Many of these genes encoding non-oxidativedecarboxylases are organized into three gene operons, encoding B, C, andD type genes. “C” type genes encode decarboxylases like AroY, and whilethe specific function of the B and D type genes are not known althoughthey are sometimes shown to be necessary for realizing full activity ofthe C type decarboxylase (Lupa et al 2005; Jimenez et al 2013; Lin et al2015; Sonoki et al 2014). Weber et al (2012) cloned the B, C and D genesfrom Klebsiella pneumoniae or Sedimentibacter hydroxybenzoicus into ahigh-copy-number yeast vector pRS4K-HKT7 and performed fermentation in amedium with externally added PCA to determine the presence of PCAdecarboxylase activities from these gene clusters; however, neither wasany effort made to determine whether the PCA decarboxylase encoded bythe C gene was dependent on the B or D gene in these gene clusters, norwas any attempt made to determine whether the expression of these geneclusters comprising B, C and D genes were able to enhance the muconicacid production using a non-aromatic carbon source such as glucose.

The gene encoding AroY that is used in specific examples disclosedherein is from Klebsiella pneumoniae, but the local gene structure ofthis aroY gene reveals no transcriptionally linked genes that wouldencode either a B or D type enzyme. Previous studies have shown thatinclusion of another B type gene, kpdB, from Klebsiella pneumoniae (partof an operon from a 4-hydroxybenzoic acid decarboxylase) can increaseAroY activity when producing muconic acid from lignin-related aromaticcompounds (Sonoki et al 2014). However, lignin is a complex mixture ofchemicals, which requires costly downstream purification processes inorder to produce a pure product such as muconic acid. Johnson et al(2016) have shown that in a Pseudomonas putida strain expressing AroY(PCA decarboxylase) from Enterobacter cloaceae, co-expression of an EcdBprotein from E. cloaceae having 89.3% sequence identity to KpdBincreased the muconic acid production using glucose as a source ofcarbon from 1.44 g/L to 4.92 g/L at the end of a 54 hour fermentation;however, the muconic acid yield in this Pseudomonas putida based systemwas still found to be extremely (0.077 mol/mol), thus making thisPseudomonas putida based system unsuitable for commercial scaleapplications. Therefore there is still a need for an improved muconicproduction process from inexpensive, purer, non-aromatic carbon sourcessuch as carbohydrates and other non-aromatic compounds (see above).Recent studies have shown that UbiX, a homolog of KpdB, produces aprenylated flavin mononucleotide cofactor and this cofactor supports thedecarboxylation activity of UbiD in ubiquinone formation (White et al2015; Payne et al 2015). Although previous characterizations of UbiX andits homologs refer to the proteins as either4-hydroxy-3-polyprenylbenzoate decarboxylases, hydroxybenzoatedecarboxylasse, subunit B proteins, phenolic acid decarboxylases, orphenylacrylic acid decarboxylases, these proteins are now moreaccurately annotated as flavin prenyltranferases.

Additional limitations in the production of muconic acid can beattributed to the limitations of the precursor metabolites PEP and E4P.Changes that eliminate or lower the consumption of these metaboliteshave been shown in the present invention to improve product formation.For instance, PEP availability has been improved by replacing the nativeglucose transport, the phosphotransferase system, (PTS) with analternate system (Glf-Glk from Zymomonas mobilis). E coli's native useof the PTS requires the utilization of PEP for transport andphosphorylation of glucose. The Glf-Glk system utilizes a facilitateddiffuser and a glucokinase plus ATP for transport and phosphorylation ofglucose, respectively. Use of Glf-Glk in place of a PTS has been shownto improve the yield and titer for several aromatic products. PEP is asubstrate for a variety of biological reactions. In addition to glucosetransport by the PTS, PEP is a substrate for pyruvate kinase, resultingin the production of ATP and pyruvate providing half of the ATPgenerated in glycolysis. Inactivation of the genes encoding for pyruvatekinase (pykA or pykF) has been demonstrated to increase yield ofproducts in the aromatic pathway (Escalante et al. 2010). PEP is alsothe substrate for the production of oxaloacetate by phosphoenolpyruvatecarboxylase (Ppc). In E. coli, this is an essential gene (Baba et al.2006), and strains deleted for ppc are unable to grow on minimal media.There are other organisms that do not have phosphoenolpyruvatecarboxylase, but instead replenish oxaloacetate from pyruvate withpyruvate carboxylase (Pyc), e.g. S. cerevisiae. The addition of pyruvatecarboxylase in E coli has been investigated, but only for theimprovement of succinate production (Lin et al. 2004; Vemuri, Eiteman,and Altman 2002) not for any aromatic products.

The specification in this patent application provides several differentaspects of invention related to the construction of a microbial strainfor efficient production of muconic acid. A person skilled in the artcan compile several different aspects of the present invention toconstruct a biocatalyst with very high efficiency for the production ofmuconic acid.

EXPERIMENTAL SECTION General Remarks

Strain and inoculum preparations: A list of the bacterial strains usedin the present invention is provided in Tables 1 and 2. A list of theplasmids used in the present invention is provided in Table 3. Allspecific examples of strain constructions disclosed herein are derivedfrom a wild type E. coli C strain (ATCC 8739), or E. coil K-12 strains(YMC9 or MM294) but the genetic elements disclosed herein can beassembled in any other suitable E. coli strain, and the expressioncassettes or appropriate analogs and homologs of the genetic elementsdisclosed herein can be assembled in any other suitable microorganism,such as other species of bacteria, archaea, yeast, algae, andfilamentous fungi that can be used for the commercial production of cis,cis-muconic acid through a fermentative process.

E. coli C is capable of fermenting 10% glucose in AM1 mineral media. AM1medium contains 2.63 g/L (NH₄)₂HPO₄, 0.87 g/L NH₄H₂PO₄, 1.5 mM MgSO₄,1.0 mM betaine, and 1.5 ml/L trace elements. The trace elements areprepared as a 1000× stock and contained the following components: 1.6g/L FeCl₃, 0.2 g/L CoCl₂.6H₂O, 0.1 g/L CuCl₂, 0.2 g/L ZnCl₂.4H₂O, 0.2g/L NaMoO₄, 0.05 g/L H₃BO₃, and 0.33 g/L MnCl₂.4H₂O. The pH of thefermentation broth is maintained at 7.0 with 1.0-10.0 M KOH or 1.0-9.0 Mammonium hydroxide.

Fermentations: Fermentations were started by streaking on a fresh NBS-2%glucose (Jantama et al., 2008a) plate from a 40% glycerol stock of E.coli strain genetically engineered and stored in a −80° C. freezer.Plasmids, if present, are retained by including the appropriateantibiotic(s) in the agar plates and liquid media. Ampicillin (sodiumsalt) is used at 150 mg/L, spectinomycin HCL at 100 mg/L, tetracyclineHCl at 15 mg/1, and kanamycin sulfate at 50 mg/l. After 24 to 48 hours(37° C.), a single colony is picked into 25 ml of the same medium in ashake flask. After shaking at 200 rpm at 37° C. until the cells havegrown to an OD₆₀₀ of about 1.0, the culture is cooled on ice and anequal volume of sterile 80% glycerol is added. 2 ml aliquots are thenfrozen at −80° C. to be used as inocula for fermentations. The term“titer” means the amount of fermentation product produced per unitvolume of fermentation fluid and the term “yield” means the ratio offermentation product produced to carbon source consumed (g/g ormol/mol).

Cell growth: Cell mass was estimated by measuring the optical density at550 nm (OD₅₅₀) or 600 nm (OD₆₀₀) using a Thermo Electronic Spectronic 20spectrophotometer.

Analysis of intermediates in shikimic acid pathway and muconic acidpathways: Total muconic acid produced in fermentation broths, whichincludes cis, cis-muconic acid and cis, trans-muconic acid, and otherbiochemical intermediates were assayed by HPLC with a Waters Allianceinstrument, and monitoring absorbance at 210 nm or refractive index at45° C., using standards purchased from Sigma-Aldrich. The column was aBioRad Aminex HPX-87H run at 50° C. with 8 mM sulfuric acid as themobile phase at a flow rate of 0.6 ml/min for 40 minutes. Achromatograph of purchased standards (Sigma-Aldrich) is shown in FIG. 4.To prepare for HPLC, fermentation samples are diluted 10 or 100 fold in0.05 M potassium phosphate buffer, pH 7.0, to preserve the cis, cis-formof muconic acid from isomerizing to the cis, trans-form.

To separate the isomers of muconic acid, the samples prepared as abovewere run in a second HPLC system. The instrument was an Agilent 1200HPLC, the column was an Agilent Eclipse XDB-C18, 4.6×150 mm run at 30degrees Centigrade with a mobile phase of 50 mM KH₂PO4 in 30% methanoladjusted to pH 3.0 with phosphoric acid. The flow rate was 1 ml/min for4 minutes, with detection by absorbance at 278 nm. The cis,trans-muconic acid standard was created by dissolving cis, cis-muconicacid in water and allowing it to undergo spontaneous acid catalyzedisomerization for about 2 hours at room temperature, until the HPLC peakhad completely shifted to a new position. The other standards werepurchased from Sigma-Aldrich. A chromatograph showing standards is shownin FIG. 5.

(094) Composition of muconic acid production medium for the fermentationprocess: Each liter of fermentation medium contains 50 ml/L of 1MKH₂PO₄, 10 ml of 200 g/L Citric acid+25 g/L Ferric citrate, 1.2 ml of98% Sulfuric acid, and a drop of Antifoam 204. These components weremixed with enough water to allow room for addition of other componentsbelow. After autoclaving, the following components were added: 10, 20,30 or 40 ml of 50% glucose (to give 5, 10, 15, or 20 g/l final), 2 ml of1M MgSO4, 1 ml of 0.1M CaCl2, 10 ml of 1000× Trace elements (Jantama etal. 2008a), and if necessary 1, 2, 4, or 8 ml of 50 g/L Phenylalanine+50g/L Tyrosine+50 g/L Tryptophan (to give 0.5, 0.1, 0.2, or 0.4 g/lfinal), 10 ml of 1 g/L p-hydroxybenzoic acid+1 g/l p-aminobenzoic acid+1g/L 2,3-dihydroxylbenzoic acid (the last three compounds are referred toas the aromatic “vitamins” or “vitamin-like componds”, and, asnecessary, 1 ml of 150 mg/ml Ampicillin (sodium salt) and/or 1 ml of 100mg/ml Spectinomycin HCl. Aromatic amino acids and vitamins were notrequired and were not used for strains expressing functional AroEprotein.

For shake flasks, NBS salts (Jantama et al. 2008a) plus 0.2 M MOPSbuffer, pH 7.4 was substituted for the pre-autoclave mix describedabove, but the glucose and other additives were the same. For fed batchfermentation, the feed bottle contained 600 g/L of anhydrous glucoseand, if necessary, 32 ml/L of 50 g/L phenylalanine+50 g/L tyrosine+50g/L tryptophan. 9 M NH₄OH was used as base to maintain the pH of thefermentation medium. Aromatic amino acids and vitamins were not requiredand were not used for strains expressing a functional AroE protein.

Fed-batch fermentations were performed in 7 L New Brunswick ScientificFermentors with pH, DO, temperature, glucose, and feed rate controlledby either DCU controllers or Biocommand Software. The medium was 50 mMK2HPO4, 20 mM K2SO4, 3 mM MgSO4 and trace elements. The trace elementsare prepared as a 50× stock and contained the following components: 1.6g/L FeCl₃, 0.1 g/L CuCl₂, 0.2 g/L ZnCl₂.4H₂O, 0.2 g/L NaMoO₄, 0.05 g/LH₃BO₃, and 0.55 g/L MnCl₂.4H₂O. The temperature was maintained at 37°C., the pH was maintained at 7.0 by 9N ammonium water. Aeration was at0.5 vvm and the dissolved oxygen (DO) was maintained at 30% byautomatically increasing the impeller's speed from 750 rpm to 1200 rpm.The initial glucose concentration in the medium was around 20 to 25 g/L.Feed glucose solution was added to the fermentor when the concentrationdropped to below 5 g/L. The initial glucose feed rate was 4 g/L/hr andwas ramped up to a feed rate of 7 g/L/hr by 48 hours after which it wasmaintained at 7 g/L/hr.

Construction of plasmids expressing muconic acid pathway genes: Thethree heterologous genes required for conversion of DHS to muconic acidwere cloned either singly or in combination into a low-copy plasmid,pCL1921 (Lerner and Inouye, 1990). The DNA sequence of pCL1921 is givenin SEQ ID No. 20 in Table 7. Briefly, the coding sequences of catAX,aroY and aroZ analogs or homologs were codon-optimized for expression inE. coli and commercially synthesized (GeneArt, Invitrogen). Thesesequences were then PCR amplified using a forward primer carrying aunique ribosome-binding site and a reverse primer carrying a uniqueterminator sequence for each gene. The resulting PCR fragment wasdigested with restriction enzymes and cloned downstream of a uniqueconstitutive promoter sequence by standard molecular cloning procedures.The promoter sequences were cloned by PCR amplification from source DNAsequences previously described (United States Patent Application20090191610; U.S. Pat. No. 7,244,593) followed by restriction digestionand standard molecular cloning. The promoter-RBS-codingsequence-terminator sequence together constituted an expressioncassette. Individual expression cassettes were next combined to generateplasmids expressing one, two or all three muconic acid pathway genes.

The present invention is further illustrated using the followingexamples; however the examples provided herein in either alone or in anycombination thereof should be construed to limit the scope or theembodiment of the invention. The claims provided at the end define thescope of the invention. A person skilled in the art can clearlyunderstand the scope of the present invention as defined the claims. Aperson skilled in the art can make modifications or changes to thetechnical solutions provided by the invention without departing from thespirit and scope of the present invention.

EXAMPLE 1 Increasing Expression of aroG and aroF Genes

The tyrR gene of E. coli can be mutated by any one of a number ofwell-known methods, such as chemical or radiation mutagenesis andscreening (for example by PCR and DNA sequencing) or selection foranalog resistance (for example, resistance to 4-fluorotyrosine),transposon mutagenesis, bacteriophage Mu mutagenesis, or transformation.In a preferred embodiment, the mutation in tyrR gene is a null mutation(a mutation that leaves no detectable activity), and in a morepreferable embodiment, at least a portion of the tyrR gene is deleted.This can be accomplished, for example, by using a two-steptransformation method using linear DNA molecules (Jantama et al, 2008a;Jantama et al, 2008b). In the first step, a cam^(R), sacB cassette isintegrated at the tyrR locus to replace most or all of tyrR open readingframe by double recombination and selecting for chloramphenicolresistance. In the second step, a linear DNA comprising a deletedversion of the tyrR gene is integrated by double recombination,selecting for resistance to 5% sucrose in a rich medium such as LB.Correct deletions are identified and confirmed by diagnostic polymerasechain reaction (PCR). The purpose of deleting tyrR is to increaseexpression of aroG and aroF. An alternative approach that achieves asimilar result is to replace the native promoter in front of aroG and/oraroF with a strong constitutive promoter and add, if necessary, atranscription terminator. More details on how this is accomplished ingeneral are given in Example 4 below.

The latter of the two approaches described above for overcoming therepression of AroG and AroF activities by TyrR protein is preferable,since deletion of tyrR can cause unwanted overexpression of genes suchas aroLM (Neidhardt and Curtiss, 1996). More detail on how this isaccomplished in general is given in Example 4 below.

EXAMPLE 2 Feedback Resistant AroG and AroF

Mutations in the aroG gene that lead to a feedback resistant AroG enzyme(3-deoxy-D-arabinoheptulosonate-7-phosphate synthase or DAHPS) are wellknown in the art (Shumilin et al, 1999; Kikuchi et al, 1997; Shumilin etal, 2002). Also well known are methods for creating, identifying, andcharacterizing such mutations (Ger et at, 1994, Hu et al., 2003). Apreferable mutation is one that leads to complete resistance toinhibition by phenylalanine. Any of the known published feedbackresistant mutations can be introduced into an aroG gene contained in thechromosome or on a plasmid by any of a number of well known methods, oneexample of which is mutagenic PCR in which the desired mutation issynthesized as part of a PCR priming oligonucleotide (Hu et al., 2003).Correct installation of the mutation is confirmed by DNA sequencing. Thesequence of the wild type aroG gene from E. coil C is given in SEQ IDNo. 18. A preferred mutation is a point mutation that changes amino acid150 of AroG from proline to leucine, for example by changing codon 150from CCA to CTA (Hu et al, 2003). In a more preferred embodiment, codon150 is changed from CCA to CTG, which is a preferred codon in E. coli.This particular allele of aroG is preferred, since the encoded DAHPsynthase is completely resistant to inhibition by phenylalanine up to 3mM, and it has a specific activity similar to the wild type enzyme (Huet al., 2003).

Additional feedback resistant aroG alleles can be obtained bymutagenesis and selection for resistance to one or more phenylalanineanalogs, such as beta-2-thienylalanine, p-fluorophenylalanine,p-chlorophenylalanine, o-fluorophenylalanine, and o-chlorophenylalanine,followed by demonstrating that the mutation causing the resistance islinked to the aroG gene (Ger et al., 1994; U.S. Pat. No. 4,681,852).Linkage to aroG can be demonstrated directly by DNA sequencing or enzymeassay in the presence and absence of phenylalanine, (Ger et al., 1994)or indirectly by phage mediated transduction and selection for a geneticmarker at or near the aroG locus that can be selected, either for oragainst (U.S. Pat. No. 4,681,852). Such a genetic marker can be adeletion or point mutation in the aroG gene itself, or a mutation in anysuitable closely linked gene such as nadA in case of E. coli. For anexample in E. coli, after mutagenesis and selection for phenylalanineanalog resistance, individual mutants or pools of mutants can be used asdonors for P1 mediated transduction into a naïve recipient that isdeleted for all three DAHP synthase genes, aroG, aroF, and aroH, andselecting for growth on an appropriate minimal medium. The transductantswill then be enriched for mutations in the desired gene(s).Alternatively, after mutagenesis and selection for analog resistance,individual mutants or pools of mutants can be used as donors for P1mediated transduction into a naïve recipient strain that contains a nullmutation in the nadA gene, again selecting for growth on an appropriateminimal medium lacking nicotinamide. Another approach is to select forresistant mutants in a strain background that contains a transposon, forexample Tn10, insertion near the aroG gene, such as in the nadA gene. P1transduction from analog resistant mutants into a strain background thatdoes not contain said transposon and selecting for tetracycline or otherappropriate antibiotic resistance will enrich for the desired aroGmutations. In all such approaches, feedback resistance is ultimatelyconfirmed by enzyme assay and DNA sequencing of the gene. We shall referto alleles of aroG that are resistant to feedback inhibition as aroG*.

Strain WM191 (ΔtyrR, ΔaroF) was derived from YMC9 (ATCC 33927). The twostep gene replacement method (Jantama et al., 2008a) was used to installclean deletions in both tyrR and aroF, to give strain WM191. Next, anadA::Tn10 allele was transduced in from CAG12147 (CGSC 7351, ColiGenetic Stock Center, Yale University) to give strain WM189 (ΔtyrR,ΔaroF, nadA:: Tn10). Selection was on LB plus tetracycline HCl (15mg/l). Strain RY890 (ΔtyrR::kan, aroF363) was derived from MM294 (ATCC33625) in three steps by P1 transduction. The donor strains, in order,were JW1316-1 (CGSC 9179, Coli Genetic Stock Center, Yale University),NK6024 (CGSC 6178, Coli Genetic Stock Center, Yale University), andAB3257 (CGSC 3257, Coli Genetic Stock Center, Yale University), and thethree selections, in order, were LB plus kanamycin sulfate (50 mg/l), LBplus tetracycline hydrochloride (15 mg/l), and NBS minimal glucose(Jantama et al., 2008a) with thiamine HCl (5 mg/l).

WM189 was mutagenized with UV light to about 20% survival and plated onNBS minimal glucose medium (Jantama et al., 2008a) containingo-fluorophenylalanine (1 mM), thiamine (5 mg/l), and nicotinamide (1mM). Colonies from each of several plates were collected into separatepools, and P1vir lysates were made on each pool. These lysates were usedto transduce WM191 to tetracycline resistance (15 mg/l) on LB medium,and the colonies obtained were replica plated to NBS minimal glucosemedium containing o-fluorophenylalanine at 1 mM, thiamine (5 mg/l), andnicotinamide (1 mM). Colony replicas that survived both tetracycline andanalog were assumed to contain a feedback resistant mutation in aroG.Eight individual colonies from 5 independent pools were chosen for DNAsequencing. The aroG coding regions were amplified by polymerase chainreaction and sequenced. The results, shown in Table 4, revealed thateach of the eight strains contained a point mutation in their aroG gene.Some of the alleles were identical to published alleles, but some werenovel.

A Plvir lysate from one of the pools described above was used totransduce RY890 (which has an aroG wild type allele) to tetracyclineresistance and resistance to o-fluorophenylalanine (0.3 mM) by replicaplating as described above. Four colonies, named RY893, RY897, RY899,and RY901, were picked for DNA sequencing (Table 4), and again, two ofthe alleles were identical to a published allele, but two were novel.Strain RY902, which is isogenic to the latter four strains, but containsa wild type aroG gene, was constructed as a control, by transductionfrom CAG12147. These five strains were grown overnight in shake flasksin 25 ml NBS minimal glucose (15 g/l) plus thiamine HCl (5 mg/l) andnicotinamide (1 mM). The resulting cells were harvested bycentrifugation, resuspended to be rinsed with 10 ml water,re-centrifuged, and resuspended in 0.5 ml of 50 mM potassium phosphate,pH 7.0. The suspended cells were lysed by vortexing with three drops ofchloroform, and the crude lysate was assayed for DAHP synthase activityusing a method similar to a method described in the literature (Hu etal., 2003), with the following modifications. The phosphate buffer was50 mM (final concentration), pH 7.0, the final erythrose-4-phosphateconcentration was 2 mM, the final phosphoenol pyruvate concentration was5 mM, the incubation temperature was 30° C., and the reaction wasstopped at 10 minutes. We define 1 mU as the activity that produced 1nMole of DAHP per minute per milligram protein. To test for feedbackresistance, each crude lysate was assayed with or without phenylalanineat a final concentration of 18 mM. The assay results are shown in Table5. The enzymes showed varying specific activity and resistance tophenylalanine, but all of selected mutant versions that were tested weresignificantly more resistant than the wild type controls.

The aroG alleles from RY893, RY899, RY901, and RY902, described above,were introduced into a muconic acid producing strain background asfollows. Plvir lysates from the aroG* and aroGwt donor strains were usedto transduce MYR219 (E. coli C, ΔaroE, Δack::P₁₅-aroB, pMG37) totetracycline HCl resistance (15 mg/l), to give new strains RY903, RY909,RY911, and RY912, respectively. Each of these strains was thentransduced to kanamycin sulfate resistance (50 mg/l) using a P1virlysate of JW1316-1, to introduce the ΔtyrR:kan allele, to give strainsRY913, RY919, RY921, and RY922, respectively. Spectinomycin selectionwas maintained throughout to maintain the muconic plasmid. The resultingfour strains were grown for 48 hours at 37° C. in shake flasks in 25 mlNBS minimal medium (Jantama et al., 2008a) containing supplements of 20g/l glucose, 0.2 M MOPS buffer, pH 7.4, nicotimamide (1 mM),phenylalanine (100 mg/l), tyrosine (100 mg/l), tryptophan (100 mg/l),p-hydroxybenzoic acid (1 mg/l), p-aminobenzoic acid (1 mg/l),2,3-dihydroxybenzoic acid (1 mg/l), phenol red (10 mg/l), and ammoniumsulfate (1 g/l). The pH was kept close to 7 as estimated by eye from thecolor of the phenol red, against a pH 7.0 standard, by manual additionof 1 ml aliquots of 1.0 M KOH as called for to the shake flasks. Themuconic acid produced was assayed by HPLC as described above, and theresults are shown in Table 6. All three strains that contain a feedbackresistant aroG* allele produced more muconic acid than the isogenicstrain containing the wild type aroG allele. In a separate experimentdisclosed herein, strain MYR205, containing aroG on multicopy plasmidpCP32AMP, produced 1.5 g/l muconic acid in a shake flask. Thus, theinventors have shown that the combination of ΔtyrR and single copychromosomal aroG* can perform well compared to an isogenic aroG plasmidcontaining strain to produce muconic acid in shake flasks. The inherentsuperior genetic stability of the chromosomal alleles compared toplasmid alleles, plus the alleviation of the need for a selective mediumto hold in a plasmid, makes the novel strains described herein moresuitable for large scale commercial fermentations. Furthermore, nochemical inducer was required for expression of muconic acid pathwaygenes. Thus, strains of the instant invention described above arcimproved over those of the prior art (Niu et al., 2002), all of whichcontain the gene for overexpression of DAHP synthase on an undesirablemulticopy plasmid.

In a similar fashion to that described above for AroG, a mutation thatleads to an AroF or AroH isozyme that is resistant to feedbackinhibition by tyrosine can be installed on a plasmid or in thechromosome. A preferred mutation is a point mutation that changes aminoacid 148 of AroF from proline to leucine, for example by changing codon148 from CCG to CTG (Weaver et al., 1990), to give a gene named aroF*.Other alleles of aroF* can be isolated by resistance to tyrosine analogs(for example o-fluorotyrosine, m-fluorotyrosine, p-fluorophenylalanine,etc.) in a fashion analogous to that described above for aroG* alleles.aroF* alleles can be selected, enriched for, and transduced by linkageto a transposon or a kanamycin resistance insertion, for example in aclosely linked ΔyfiR::kan as in a strain such as JW2584 (CGSC 10051,Coli Genetic Stock Center, Yale University).

EXAMPLE 3 Deletion of aroE from Chromosomal DNA and Muconic AcidProduction

In this example the effect of overexpression of aroB and aroG onmulticopy plasmids as well as the expression of genes coding forproteins functional in the muconic acid pathway was investigated. StrainMYR34 containing a deletion in the aroE gene coding for shikimatedehydrogenase was used as parent strain in these studies. The deletionof chromosomal copy of aroE was accomplished in a fashion similar tothat described above in Example 1. When MYR34 was transformed with theplasmid pCP32AMP overexpressing the aroG gene coding for DAHP synthaseprotein functional in the shikimic acid pathway, there was a significantincrease in the accumulation of DHS. When MYR34 was transformed with theplasmid expressing aroB from a constitutive promoter, no significantincrease in the accumulation of DHS was noticed. However, when the E.coli strain MYR34 was transformed with the plasmid expressing both aroBand aroG genes, there was an increase in the accumulation of DHS thanobserved with MYR34 transformed with aroG alone thus suggesting aroB asa secondary bottleneck in DHS production (FIG. 6).

In the experiments presented in FIG. 7, the effect of an additional copyof the aroB gene integrated into the host chromosomal DNA was examined.In the E. coli strain MYR170 derived from MYR34, an additional copy ofthe aroB gene under the control of the P₁₅ promoter was integrated intothe host chromosome at the ack locus. When MYR170 strain was transformedwith the pCP32AMP plasmid, there was a slight increase in the DHSaccumulation when compared to the DHS accumulation detected in the MYR34strain transformed with the same plasmid. This slight increase in theaccumulation of DHS in the MYR170 can be attributed to an additionalcopy of aroB gene integrated into the host chromosomal DNA. When MYR170was transformed with pCP54 expressing both aroB and aroG genes, therewas a further increase in the DHS accumulation suggesting aroB as asecondary bottleneck in DHS production.

FIG. 8 provides the results on muconic acid production with the E. colistrains MYR34 and MYR170. Having established that in the aroE deletionstrains MYR34 and MYR170, with overexpression of aroB and aroG genesthere is an accumulation of DHS, efforts were made to see whether theexpression of “muconic pathway” genes coding for the proteins functionalin muconic acid production pathway would lead to conversion of DHS intocis, cis-muconic acid. In these experiments, the E. coli stains MYR34and MYR170 were transformed either with the plasmid pMG37 alone or withboth plasmids pMG37 and pCP32AMP. The plasmid pMG37 expresses aroZ, aroYand catAX genes coding for proteins functional in muconic acid pathway.The =conic acid production in both MYR34 and MYR170 increased when thesebacterial strains were transformed with both the plasmids pCP32AMP andpMG37 when compared to the muconic acid production in these two strainstransformed only with pMG37 plasmid suggesting that in these strainsaraB expression is the bottleneck for cis, cis-muconic acid production.

EXAMPLE 4 Overexpression of tktA

Transketolase encoded by tktA is a key enzyme in the pentose phosphatepathway and is thought to be limiting for the production oferythrose-4-phosphate, one of the key intermediates in the production ofmuconic acid. Overexpression of tktA, which encodes transketolase, byinstalling the gene with its native promoter on a multicopy plasmid(Sprenger et al, 1995, 1995a), is known to improve flux into thearomatic pathway (Draths et al., 1992). However, such plasmids areunstable, and often require antibiotic selection for maintenance.Another approach in the prior art was to add one additional copy of thetktA gene to the chromosome of the host strain (Niu et al., 2002).However, one additional copy of tktA with its native promoter is notsufficient to saturate the aromatic pathway with erythrose-4-phosphate,since its native promoter is not very close to the ideal. As such, theprocess needs substantial improvement.

Improved overexpression of tktA can be obtained, for example, bysubstituting the native tktA promoter in the chromosome with a strongconstitutive promoter, for example a P₁₅ or P₂₆ promoter from Bacillussubtilis phage SPO1 (SEQ ID No. 1 and SEQ ID No. 2, respectively), orthe P_(R) promoter from bacteriophage lambda (SEQ ID No. 3). This isaccomplished in two steps as described in Example 1, except that thecam^(R), sacB cassette is used to replace the native chromosomal tktApromoter in the first step. In the second step, the strong constitutivepromoter is installed by transforming with a linear DNA comprising thestrong constitutive promoter, flanked by at least 50 bases of the 5′ endof the tktA coding region on the downstream side and at least 50 basepairs of homology just upstream of the native tktA promoter on theupstream side of the strong constitutive promoter, and selecting forsucrose resistance. Improved expression from such an expression cassetteis also accomplished by increasing the stability of the mRNA that istranscribed from the expression cassette. Improvement of the mRNAstability is accomplished by adding a stem loop structure at either the5′ end of the mRNA, the 3′ end of the mRNA, or both. A stem-loopstructure is often present at the end of an mRNA that is naturallyterminated by a rho-independent transcription terminator, but if it isnot, then a rho-independent transcription terminator can be added to theDNA sequence by well known methods of genetic engineering (ligation,PCR, etc.). Such a terminator can be comprised of an inverted repeat ofbetween 4 and 20 bases in each repeat, separated by a “loop” of 3 ormore bases, and followed by a region of one or more bases that isenriched for T's (deoxythymidine). The inverted repeats are rich in G'sand C's (deoxyguanidine and deoxycytidine). Similarly, a stem-loop canbe constructed into the 5′ end of an mRNA by inserting a DNA sequencejust downstream from the start point of transcription, but before theribosome binding site, that contains a stem-loop as described above, butwithout the T-enriched region. An example of this is given inassociation with the P₁₅ promoter (SEQ ID No. 1).

In the analysis of the effect of overexpression of the tktA gene on theflow of carbon through the shikimic acid pathway, E. coli strain MYR170was used as a parental strain. MYR170 has a deletion in the aroE genecoding for shikimate dehydrogenase enzyme and an additional copy of thearoB gene at the ack locus.

In the experiments described in the FIGS. 9, 10 and 11 two differentplasmids namely pCP32AMP and pCP50 were used. The plasmid pCP32AMPexpresses only the DAHP synthase aroG gene from its native promoter andthe plasmid pCP50 expresses the transketolase gene tktA from its nativepromoter along with aroG gene. MYR170, having an aroE deletion and anadditional copy of aroB gene under the control of P₁₅ promoterintegrated at the ack locus of the chromosomal DNA, was transformedindividually with pCP32AMP and pCP50 plasmids. As shown in FIG. 8 theDHS accumulation was increased further with the expression of aroG genealong with tktA gene when compared to the E. coli cells expressing onlyaroG gene.

FIG. 10 provides data on the DHS yield in two different strains namelyMYR34, MYR170 transformed with the plasmid pCP32AMP or pCP50. MYR34strain having aroE gene deletion yielded 0.1 gram of DHS per gram ofglucose consumed. The DHS yield in the MYR34 increased to 0.15 gram ofDHS per gram of glucose consumed when this strain was transformed withthe pCP32AMP plasmid with aroG gene overexpression. MYR170 has anadditional copy of aroB gene inserted at the ack locus. As a result ofthe presence of this additional copy of the aroB gene, the yield for DHSproduction in the MYR170 strain transformed with pCP32AMP was slightlyhigher than the DHS yield noted in the MYR34 strain transformed withpCP32AMP. Thus the presence of an additional copy of aroB in MYR170caused an increased carbon flow through shikimic acid pathway. Furtherincrease in the DHS yield was observed when the MYR170 strain wastransformed with plasmid pCP50 expressing both aroG and tktA genes. Thusthe presence of additional copy of tktA accounted for an increase carbonflow through shikimic acid pathway. More specifically, the effect ofpresence of additional aroB and tktA genes caused an additive effect onDHS yield.

MYR261 used in the experiments described in FIG. 11 was engineered tointegrate an additional copy of tktA gene into the chromosomal DNA ofMYR170 at the poxB locus. The desired gene replacement (poxB::tktA) inthe MYR261 strain was confirmed via PCR. MYR261 was transformed eitherwith pCP32AMP (aroG overexpression) plasmid or pCP50 (aroG and tktA overexpression) plasmid. As a control, MYR170 was transformed with pCP32AMPplasmid. As the result shown in FIG. 11 indicate, the presence of anadditional copy of tktA gene in the chromosomal DNA of MYR261 increasedthe titer for DHS production with pCP32AMP plasmid when compared to thetiter for DHS production observed in the MYR170 strain transformed withthe same plasmid. Further increase in the transketolase level in theMYR261 strain when transformed with the plasmid pCP50 over expressingtransketolase led to further increase in the titer for DHS production.The enzyme encoded by poxB, PoxB, or pyruvate oxidase, produces acetateas a reaction product. As such, the deletion of poxB that results fromthe insertion of tktA as described herein removes a potentially activepathway for acetate production. Similarly, simultaneous insertion ofP₁₅aroB and deletion of ackA, which encodes AckA, or acetate kinase, asdescribed below in Example 12 below, removes another potentially activepathway to acetate. Production of acetate is generally undesirable infermentations (Jantama et al., 2008b). As such, these deletions can beuseful for reducing acetate production.

FIG. 12 provides the titer for muconic acid and acetic acid productionin MYR170, MYR261 and MYR305 strains of E. coli after transformationwith the plasmids pCP32AMP and pMG37. MYR305 is derived from MYR170 bymeans of deleting poxB gene from the chromosomal DNA while MYR261 is aMYR170 derivative wherein the poxB gene has been inactivated by means ofinserting an additional copy of the tktA gene. As mentioned above, theplasmid pCP32AMP expresses the aroG gene coding for DAHP synthaseprotein functioning in the shikimic acid biosynthetic pathway leading tothe accumulation of DHS due to the deletion of aroE gene in the E. colistrains MYR170, MYR261 and MYR305. With the expression of muconicpathway genes namely aroZ, aroY and catAX on the plasmid pMG37, the DHSis converted into cis, cis-muconic acid as illustrated in FIG. 2. Withthe presence of an additional copy of the aroB gene and the tktA gene inthe MYR261 strain, there was a slight increase in the production ofmuconic acid accompanied by a decrease in the accumulation of aceticacid.

EXAMPLE 5 Overexpression of talA or talB

The talB gene encodes the predominant transaldolase in E. coli, but thetalA gene also encodes a minor transaldolase. Overproduction oftransaldolase is known to improve flux into the aromatic pathway (Lu andLiao, 1997; Sprenger, 1995; Sprenger et al, 1995b). In the prior art,this was accomplished by overexpression of the tal gene (now known to bethe talB gene) on a multicopy plasmid from its native promoter (Lu etal., 1997, Sprenger et al., 1995b). However, such plasmids are unstable,and require antibiotic selection for maintenance. Thus, there is a needfor an improved process. Improved expression of talB can be obtained,for example, by substituting the native talB promoter in the chromosomewith a strong constitutive promoter, for example a P₁₅ or P₂₆ promoterfrom Bacillus subtilis phage SPO1 (SEQ ID No. 1 and SEQ ID No. 2,respectively), or the P_(R) promoter from bacteriophage lambda (SEQ IDNo. 3). This is accomplished in two steps as described in Example 1,except that the cam^(R), sacB cassette is used to replace the nativechromosomal talB promoter in the first step. In the second step, thestrong constitutive promoter is installed by transforming with a linearDNA comprising the strong constitutive promoter, flanked by at least 50bases of the 5′ end of the talB coding region on the downstream side andat least 50 base pairs of homology just upstream of the native talBpromoter on the upstream side of the strong constitutive promoter, andselecting for sucrose resistance. The talA gene can also beoverexpressed by a similar method, but it is preferred to over expressthe talB gene, since it encodes the predominant activity (Sprenger,1995; Sprenger et al, 1995b). See Example 4 for more details onconstruction of the expression cassette designed for overexpression.

EXAMPLE 6 Expression of aroZ, aro and catAX Genes

To demonstrate conversion of endogenous DHS produced by E. coli intomuconic acid, heterologous genes catAX from Acinetobacter sp. ADP1, aroYfrom Klebsiella pneumoniae, and quiC from Acinetobacter sp. ADP1, werecloned under strong constitutive promoters (P₁₅, P_(R), and P_(26,)respectively) in a low-copy plasmid, pCL1921 (Lerner and Inouye, 1990)to generate a ‘muconic plasmid’ pMG37. MYR34 strain derivatives carryingthe empty vector (pCL1921) or pMG37 were grown at 37° C. for 17 hrs. ina shake flask medium (NBS minimal medium supplemented with aromaticamino acids and vitamins) containing 2% glucose. Supernatants werecollected and analyzed by HPLC. In contrast to MYR34/pCL1921 which showsaccumulation of DHS, MYR34/pMG37 shows production of muconic acid (FIG.13). No significant amount of DHS, or intermediate products such as PCAand catechol were detected from the latter strain, suggesting that theheterologous genes expressed from pMG37 were functional and sufficient.

EXAMPLE 7 Comparison of aroZ Homologs

Three different aroZ homologs and analogs were compared (FIG. 14) fortheir ability to divert DHS into the muconic acid production pathway.quiC from Acinetobacter sp. ADP1, asbF from Bacillus thuringiensis, andqa-4 from Neurospora crasser, are reported to encode for proteins thathave AroZ-like activity (Elsemore and Ornston, 1995; Fox et al, 1995;Rutledge, 1984). Each of these genes was codon-optimized for expressionin E. coli and synthesized by GeneArt (Invitrogen), and cloned under astrong constitutive P₂₆ promoter in low-copy ‘muconic plasmid’ whichalso expressed catAX and aroY genes from the P₁₅ and P_(R) promoters,respectively. MYR34/pCL1921, MYR34/pMG37 (muconic plasmid with quiC asaroZ), MYR34/pMG47 (muconic plasmid with asbF as aroZ), and MYR34/pMG70(muconic plasmid with qa-4 as aroZ) were grown at 37° C. for 48 hrs. ina shake flasks with minimal medium containing 2% glucose, the aromaticamino acids and aromatic vitamins. Supernatants were collected andanalyzed by HPLC. As expected, empty vector transformed MYR34accumulated DHS and produced no muconic acid. The two aroZ homologs andthe one analog examined were functional in diverting DHS towards muconicacid production, but to a varying degree. The MYR34 derivativeexpressing quiC gene was most robust and showed nearly 100% conversionof DHS to muconic acid with insignificant amount of DHS retention. TheMYR34 derivative expressing the fungal aroZ homologue, qa-4, followedclose with about 80% conversion of DHS to muconic acid and 20% DHSretention. Lastly, the MYR34 derivative expressing ashF gene showed only50% conversion of DHS to muconic acid and 50% DHS retention. Takentogether, under our shake flask assay conditions, the expression and/oractivity of quiC gene appeared to be the highest compared to that ofother aroZ homologs.

EXAMPLE 8 Chromosomal Integration of catAX, aroY and quiC

Muconic acid can be produced by strains that contain only chromosomallyintegrated single copies of catA-X, aroY and quiC expressed fromconstitutive promoters at adhE locus.

MYR170 (ΔaroE, Δack::P₁₅-aroB), a high DHS producer, was the host strainused for integrating the muconic acid pathway genes at the adhE locus inthe chromosome (SEQ ID No. 41). The resulting strain MYR352 wastransformed with plasmids YEp24 (medium-copy, empty vector), pCP32AMP(medium-copy, aroG expressed from native promoter), or pCP50(medium-copy, aroG and tktA expressed from their respective nativepromoters) to generate derivative strains. The latter two plasmids wereused to increase DHS production. Strains were grown at 37° C. for 72hrs. in shake flask medium containing 2% glucose as described above.Supernatants were collected at 72 hrs. and analyzed by HPLC. Asexpected, the aroG and aroG/tktA transformed MYR352 derivatives showedan overall increase in total product formation compared to an emptyvector control (FIG. 15). All of the MYR352 transformants producedmeasurable titers of muconic acid, demonstrating for the first time thatmuconic acid can be produced by a strain that contains only integrated“muconic pathway” genes and without a fed chemical inducer of geneexpression.

Not all DHS that was produced in any of these MYR352 derivative strainswas converted to the end product muconic acid. Instead, there was asignificant amount of catechol accumulation (FIG. 15), suggesting thatexpression or activity of catAX is limiting when it is expressed from asingle copy on chromosome. Since the major accumulating intermediate wascatechol, it is likely that quiC and aroY gene expression and/oractivity is sufficient in the MYR352 strain background for muconic acidsynthesis.

The MYR352 strain derivatives were compared in parallel with analogousMYR219 strain derivatives. MYR219 strain is same as MYR170 strain butcontains low-copy plasmid pMG37 expressing muconic acid pathway genes.Thus, the main difference between MYR352 and MYR219 strains is withreference to the dosage of muconic acid pathway genes (1 copy vs. about5 copies, respectively). In contrast to MYR352 derivative strains,MYR219 derivative strains showed very little accumulation of catechol orother intermediates, and successfully produced the end product muconicacid. Together, these results indicate the need for increasing catAXactivity in strains such as MYR352.

EXAMPLE 9 Expression of catAX

Accumulation of catechol and inefficient production of muconic acid inMYR352 strain is due to limiting dosage and/or activity of the catAXgene product(s). As described above, MYR352 contains ΔaroE,Δack::P₁₅-aroB and chromosomally integrated single copies of catAX, aroYand quiC genes under strong constitutive promoters. This strain wastransformed with medium-copy empty vector control (YEp24) or aroG/tktAexpression plasmid (pCP50) to increase carbon flow into the aromaticamino acid synthesis pathway and produce high amounts of DHS. Growth oftransformed strains at 37° C. for 72 hrs in shake flask mediumsupplemented with 2% glucose as described above resulted in accumulationof catechol intermediate. This result suggested that catAX activity maybe insufficient in MYR352. To confirm this hypothesis, the ability ofone or more muconic acid pathway genes expressed from low-copy plasmidto alleviate catechol accumulation in MYR352/pCP50 was tested (FIG. 16).Specifically, MYR352/pCP50 was further transformed with low-copy emptyvector control (pCL1921) or plasmids expressing all three genes, twogenes, or one gene of the muconic acid production pathway. Thederivative strains were assayed in a shake flask experiment as describedabove. While increasing the dosage of aroY alone (from pMG27) or quiCalone (from pMG39) did not alleviate catechol accumulation, expressionof all of the muconic acid pathway genes (from pMG37) or catAX and aroYtogether (from pMG33), resulted in successful conversion of catechol tomuconic acid. Further, expression of catAX alone (from pMG31) wassufficient for production of muconic acid and preventing accumulation ofcatechol.

EXAMPLE 10 Constructing a Leaky aroE Mutation

In the prior art process for producing cis, cis-muconic acid, the hoststrain contains a mutation in the aroE gene named aroE353, which is anull mutation. As a result, the strain requires the feeding of thearomatic amino acids (phenylalanine, tyrosine, and tryptophan) andaromatic vitamins made from the shikimate pathway (p-hydroxy benzoicacid, p-amino benzoic acid, and 2,3-dihydroxy benzoic acid). Thearomatic amino acids arc too expensive to be fed in a commerciallyattractive process. As such, the prior art process needs a substantialimprovement. This can be accomplished by installing a leaky version ofthe aroE gene, that we shall call aroE*. Leaky mutations are obtained byfirst generating a missense mutation that changes one amino acid in thearoE coding sequence that results in a null phenotype. This can beaccomplished by any form of mutagenesis and screening for simultaneousauxotrophy for the six aromatic compounds listed above. A preferredmethod is to create a pool of mutant aroE genes by error-prone PCRmutagenesis, using Taq DNA polymerase, using wild type E. coli C genomicDNA as the template, and using PCR oligonucleotide primers thathybridize about 1000 base pairs upstream and 1000 base pairs downstreamof the aroE coding region. The resulting pool of linear DNA molecules isused to transform an E. coli C derivative that produces cis, cis-muconicacid, and which contains an integrated cam^(R), sacB cassette that hasreplaced the aroE coding region (see Example 4 for a related example),and selecting for sucrose resistance. The transformants are thenscreened for auxotrophs that have lost chloramphenicol resistance andrequire the six aromatic compounds listed above. Several independentauxotrophs are picked and tested for revertability by plating about 10⁷,10⁸, or 10⁹ cells (rinsed in minimal glucose medium) on a minimalglucose plate without the six aromatic compounds. Revertants that giverise to colonies on the plates are picked and tested for production ofcis, cis-muconic acid, but without production of substantial levels ofaromatic amino acids. Among such revertants will be strains that carryone or more mutations in the aroE gene, such that the AroE enzymeprovides enough aromatic amino acids and vitamins for growth, but not asurplus of these aromatic compounds. Another method to obtain a leakyaroE mutant is to install one of the classical revertable aroE mutants,such as aroE353 and aroE24 (both available from the Coli Genetic StockCenter at Yale University, New Haven, Conn., USA), into a cis,cis-muconic acid producing strain, and select for revertants asdescribed above.

EXAMPLE 11 Import of Glucose by Facilitated Diffusion

One of the substrates in the first committed step of the aromaticpathway is phosphoenolpyruvate (PEP). PEP is also the source ofphosphate and energy for importing glucose and some other sugars by thebacterial phosphotransferase system (PTS). Thus, when a bacterium isgrowing on a PTS-dependent sugar, there is competition between the PTSand the aromatic pathway for PEP. As such, a significant improvement inincreasing flux to the aromatic pathway can be achieved by deleting thePTS and providing an alternative pathway for sugar uptake. One solutionto this problem is to replace the PTS with the E. coli GalP permease, aproton symporter that works reasonably well for glucose uptake (U.S.Pat. No. 6,692,794). However, the proton symporter still uses energy tomaintain the proton gradient that is necessary to drive the permease. Assuch, there is a need for even further improvement in the process.

Some sugars, such as xylose, can be imported by a transporter proteinthat derives energy from hydrolysis of ATP (adenosine triphosphate).Once again, if the energy-dependent transporter can be replaced by atransporter that requires less energy, then an improvement can be made,since the energy inherent in the ATP can be conserved for otherbeneficial uses.

A significant improvement can be obtained by using a facilitateddiffusion transporter, which expends no energy for the importation ofthe sugar (Parker et al, 1995; Snoep et al, 1994). For example, theglucose facilitator from Zymomonas mobilis, encoded by the glf gene, canbe used in place of, or in addition to, the PTS in 3-dehydroshikimate(DHS) producing strains (Yi et al., 2003). However, these strains stillrely at least partly on GalP for glucose import. Since GalP requiresenergy in the form of a proton gradient for importation of glucose,there is a need for improvements in the efficiency of glucose import formuconic producing strains.

A cassette for expression of glf plus a glucokinase gene, glk, also fromZ. mobilis, can be assembled with a strong constitutive promoter, forexample P₂₆. This cassette can then be integrated into the genome of ahost strain at a location that will not interfere with production of thedesired compound, which in this case is cis, cis-muconic acid. Anexample of such a location in the E. coli chromosome is the threoninedegradation operon, tdcABCDEFG. If the growth medium contains nothreonine, then this operon is not needed or expressed, so an insertionof an expression cassette in that operon does not interfere withmetabolism.

To achieve the above described improvement, one or more of the genesencoding a PTS function are deleted, using a method similar to thatdisclosed in Example 1. For example, one or more of ptsH, ptsl, crr, orptsG can be deleted. Next galP is deleted using the process as decribedin the U.S. Pat. No. 8,871,489. The P₂₆-glf; glk cassette can then beinstalled in two steps, similar to those described in Example 1. In thefirst step, a cam^(R), sacB cassette is integrated at the tdc operon,using a linear DNA derived from pAC21 (SEQ ID No. 15), and selecting forchloramphenicol (30 mg/l) resistance. In the second step, the P₂₆-glf;glk cassette is integrated at the tdc operon, using a linear DNA derivedfrom pAC19 (SEQ ID No. 15), selecting for sucrose resistance andscreening for chloramphenicol sensitivity, and in this case, improvedgrowth on minimal glucose medium.

To test whether facilitated diffusion of glucose could substitute forthe conventional glucose import systems in E. coli, the ptsHI genes andthe galP gene were deleted from MYR34 (AaroE), and then the P₂₆-glf, glkcassette was integrated at the tdc operon, using a linear DNA derivedfrom pAC19 (SEQ ID No. 15), to give strain MYR217. MYR217 growsreasonably well on a minimal glucose medium supplemented with therequired three aromatic amino acids and three aromatic vitamin-likecompounds (FIG. 17). However, strain MYR31, which contains deletions ofptsHI and galP, but does not contain the glf, glk cassette did not showany measurable growth (FIG. 17). Thus, facilitated diffusion issufficient to replace the two conventional glucose import systems in ourstrain background.

To test whether facilitated diffusion is useful for producing compoundsderived from the aromatic pathway, MYR34 and MYR217 were transformedwith pCP54 (aroG, aroB) and pCP55 (aroG, aroB, tktA). Production of thearomatic intermediate 3-dehydroshikimate (DES) in shake flasks wascompared for these two strains (FIG. 18). With either pCP54 or pCP55,the strain using facilitated diffusion produced as much or more DHS thanthe strains using the conventional glucose import systems. Production ofDES is a good proxy for muconic acid production in engineered E. colistrains, so we can conclude that facilitated diffusion of glucose is auseful improvement for muconic acid production.

EXAMPLE 12 Overexpression of the aroB Gene

Expression of the aroB gene is reported to be rate limiting for cis,cis-muconic acid production (Niu et al., 2002). In the prior art, thiswas allegedly solved by integrating a second copy of the aroB gene withits native promoter. However, this is insufficient to alleviate the aroBlimitation, since the native promoter and ribosome binding site of thearoB gene are far from ideal. As such, the process needs substantialimprovement.

Improved overexpression of aroB can be obtained, for example, byreplacing the native aroB promoter in the chromosome with a strongconstitutive promoter, for example a P₁₅ or P₂₆ promoter from Bacillussubtilis phage SPO1 (SEQ ID No. 1 and SEQ ID No. 2, respectively), orthe P_(R) promoter from bacteriophage lambda (SEQ ID No. 3). This isaccomplished in two steps as described in Example 4, except that thecam^(R), sacB cassette is used to replace the native chromosomal aroBpromoter and/or ribosome binding site in the first step. In the secondstep, the strong constitutive promoter is installed by transforming witha linear DNA comprising the strong constitutive promoter, followed by aribosome binding site and at least 50 bases from the 5′ end of the aroBcoding sequence, including the ATG start codon, on the downstream side,and at least 50 base pairs of homology just upstream of the native aroBpromoter on the upstream side of the strong constitutive promoter, andselecting for sucrose resistance. In addition to, or instead of,installing a stronger promoter, using a similar method, a strongerribosome binding site, for example, AGGAGG, can be installed about 4 to10 base pairs upstream of the ATG translation start codon of aroB. Acopy of such a synthetic cassette, for example, a P₁₅-aroB cassette, canbe integrated in the chromosome at a locus distinct from the native aroBlocus, for example at the ack locus. Simultaneous deletion of the ackgene, as well as deleting the poxB gene as in Example 4 can help toreduce formation of unwanted acetate during fermentations.

EXAMPLE 13 Decreasing Flux through the Oxidative Branch of the PentosePhosphate Pathway

The erythrose-4-phosphate that is needed for the first committed step inthe aromatic pathway is derived from the non-oxidative portion of thepentose phosphate pathway (PPP). There are two different pathways bywhich carbon can enter the PPP. The first is from glucose-6-phosphate bythe enzymes glucose-6-phosphate dehydrogenase (encoded by the zwf gene),6-phosphogluconolactonase (encoded by the pgl gene), and6-phophogluconate dehydrogenase (encoded by the gnd gene), to giveribulose-5-phosphate. In the last of these three steps, one carbon islost as CO₂. This path into the PPP is called the oxidative branch ofthe PPP. Ribulose-5-phosphate is then converted into a variety of othersugar phosphates by the action of isomerases, epimerases, transketolase,and transaldolase. This group of reversible reactions, starting withribulose-5-phosphate, is called the non-oxidative branch of the PPP. Thesecond path by which carbon can enter the PPP is throughfructose-6-phosphate and glyceraldehye-3-phosphate (both of which comefrom the Embden-Myerhoff pathway, also known as glycolysis), which arecombined and rearranged by transaldolase and transketolase to give thevariety of other sugar phosphates, one of which iserythrose-4-phosphate. If carbon enters the PPP through this secondroute, then no CO₂ is lost. In order to improve the yield of cis,cis-muconic acid from glucose, the loss of CO₂ can be prevented byblocking the oxidative branch of the PPP, such that all carbon enteringthe PPP must come through a non-oxidative route fromfructose-6-phosphate and glyceraldehye-3-phosphate. The blocking of theoxidative branch of the PPP is accomplished by deleting the zwf gene,using a two-step method similar to that disclosed in Example 1 fordeleting the tyrR gene.

EXAMPLE 14 Increasing the Flux to and through PEP to the AromaticPathway

It is desirable to ensure that PEP is not a rate limiting intermediateon the pathway to cis, cis-muconic acid. This is accomplished, forexample, by increasing the recycling of pyruvate to PEP by the enzymePEP synthetase, which is accomplished by integrating an overexpressioncassette of the pps gene as described above in other examples. Anotherapproach is to limit the consumption of PEP by pyruvate kinase, which inE. coli is encoded by the pykA and pykF genes. In this case, theapproach is to decrease the activity of the enzyme(s). This isaccomplished by deleting one or more genes that encode pyruvate kinase(as described in Example 1 for tyrR and in the U.S. Pat. No. 9,017,976),or reducing the strength of expression of one or more of these genes,for example, by mutating the promoter, ribosome binding site, or codingsequence, such that the level of pyruvate kinase activity is decreased.For example, the RBS in front of the E. coli pykA gene is5′CGGAGTATTACATG. The ATG translation start codon is underlined. Thissequence can be mutated to CaGAGTATTACATG, CaaAGTATTACATG,CaatGTATTACATG, CaataTATTACATG, and so on, such that the RBS sequence ismade less like the consensus RBS of AGGAGG by one base change at a time.Each mutated version is then introduced into the chromosome at the pykAlocus, replacing the wild type, and cis, cis-muconic acid productionlevels are measured for improvement.

EXAMPLE 15 Conferring Growth on Sucrose

Strains derived from E. coli C do not grow on sucrose as a sole carbonsource. However, they can be genetically engineered to do so asdisclosed in the International Patent Application Publication No.WO2012/082720 and US Patent Application Publication No. US2013/0337519which are hereby incorporated by reference in its entirety. As such, acis, cis-muconic acid producing strain can be engineered to grow onsucrose as disclosed in the above mentioned application.

EXAMPLE 16 An Improved Producer of cis, cis-muconic Acid

All of the features described in Examples 1-15 can be combined in onestrain of E. coli by installing the features one after another. Theresulting strain comprises an improved cis, cis-muconic acid producer.The resulting strain can then be even further improved by integrating asecond copy of each overexpression cassette described above, one at atime, at a location separate from the location of the first copy. Anexample of a convenient and safe location is at a BsrB1 restriction sitejust downstream from the terminator of rrfF, which encodes a ribosomalRNA. The desired cassette is ligated as a blunt linear DNA into theunique BsrB1 site of plasmid pMH17F (SEQ ID No. 17). An example is theligation of the catAX expression cassette to give a plasmid namedpcatAX. In parallel, a cam^(R), sacB cassette is ligated as a bluntfragment into pMH17F to give pMH28F (SEQ ID No. 19). A linear DNAderived from pMH28 by PCR or by restriction enzyme cutting is used todeposit the cane, sacB cassette at the rrfF site. Next, a linear DNAderived from pcatAX by PCR or by restriction enzyme cutting is used toinstall the second copy of the catAX cassette at the rrfF locus, usingselection on sucrose. The resulting strain is then compared with itsgrandparent strain for cis, cis-muconic acid production to determinethat catAX was a limiting step. By a similar method, each cassette fromExamples 2-15 is tested for a rate limiting step. If a step is found tobe rate limiting, then one or more additional copies of the relevantcassette is/are integrated at yet other appropriate locations in thechromosome, leading to still further improvements in cis, cis-muconicacid production, without the need for plasmids or inducible promoters.

EXAMPLE 17 Production of cis, cis-muconic Acid by Fermentation

Cis, cis-muconic acid can be produced by genetically engineeredmicroorganisms disclosed in the above Examples 1-15. The growth mediumcan vary widely and can be any medium that supports adequate growth ofthe microorganism. A preferred medium is a minimal medium containingmineral salts and a non-aromatic carbon source, such as glucose, xylose,lactose, glycerol, acetate, arabinose, galactose, mannose, maltose, orsucrose (see above for an example of a preferred minimal growth medium).For each combination of engineered microorganism and growth medium,appropriate conditions for producing cis, cis-muconic acid aredetermined by routine experiments in which fermentation parameters aresystematically varied, such as temperature, pH, aeration rate, andcompound or compounds used to maintain pH. As cis, cis-muconic acid isproduced, one or more compounds must be fed into the fermentor toprevent pH from going too low. Preferred compounds for neutralizing theacid include alkaline salts such as oxides, hydroxides, carbonates, andbicarbonates of ammonium, sodium, potassium, calcium, magnesium, or acombination two or more of such alkaline salts.

Muconic acid production by MYR428 strain of E. coli in a 7 Literfermentor is shown in FIG. 19. MYR261 strain of E. coli with a genotypeof ΔaroE ΔackA::P₁₅-aroB ΔpoxB::tktA was transformed with the plasmidspCP32AMP and pMG37 to generate MYR428. MYR428 was grown in a 7 literfermentor as described above with glucose feeding for 48 hours. Thefinal muconic acid titer was 16 g/l (see FIG. 19).

After fermentation is complete, cells are removed by flocculation,centrifugation, and/or filtration, and the cis, cis-muconic acid is thenpurified from the clarified broth by a combination of one or moresubsequent steps, for example precipitation, crystallization,electrodialysis, chromatography (ion exchange, hydrophobic affinity,and/or size based), microfiltration, nanofiltration, reverse osmosis,and evaporation.

EXAMPLE 18 Improvement of 3,4-dihydroxybenzoic acid (PCA) decarboxylase(AroY) Activity

E. coil strain MYR993 with the genotype as provided in Table 2 was useda parental strain to generate strains with the deletion in either theubiX gene or the ubiD gene. In constructing the E. coli strains with thedeletion in ubiX a kanamycin resistance cassette was amplified usingprimers MS608 and MS609 having 45 bp of homology to each end of the ubiXgene. In constructing the E. coli strains with the deletion in ubiD, akanaymcin resistance cassette was amplified using primers MS604 andMS605 having 45 bp of homology to each end of the ubiD gene. The PCRproducts were column purified (QIAquick PCR Purification Kit, Qiagen)and used to transform the E. coli strain MYR993 (Table 2) usingpreviously developed methods (Datsenko K A, Wanner B L (2000) One-stepinactivation of chromosomal genes in Escherichia coli K-12 using PCRproducts. Proc Natl Acad Sci USA 97: 6640-6645). to produce the E. coilstains with a deletion in either ubiX gene or ubiD gene (Table 2—MYR993AubiX and MYR993 ΔubiD). The deletion stains are expected to be impairedin respiration so glucose was added to LB selection plates to providefor fermentative growth.

The E. coli strains MYR993, MYR993 ΔubiX and MYR993 ΔubiD were grown as25 ml cultures in 250 ml shake flasks at 250 rpm at 37° C. for 48 hoursin a medium comprised of 5 g K₂HPO₄, 3.5 g KH₂HPO₄, 3.5 g (NH4)₂HPO₄, 1mM MgSO₄, 0.1 mM CaCl₂, trace elements (1.6 mg FeCl₃.6H₂O, 0.2mgCoCl₂.6H₂O, 0.1 mg CuCl₂.2H₂O, 0.2 mg ZnCl₂, 0.2 mg Na₂MsO₄.2H₂O, 0.05mg H₃BO₃, 0.55 mg MnCl₂.4H₂O), and 0.2 M MOPS buffer (all per liter),using glucose as a carbon source. At the end of 48 hours of growth, theculture supernatants were analyzed for muconic acid and PCA content. Asthe results shown in FIG. 20 indicates, the parental strain MYR993accumulated primarily muconic acid in the culture medium with verylittle PCA while the E. coli strain MYR993AubiX accumulated only PCA andmuconic acid was not detectable. On the other hand, the E. coil strainMYR993ΔubiD showed the reduced accumulation of both muconic acid andPCA. The conclusion was that UbiX protein is needed for AroY (PCAdecarboxylase) activity.

EXAMPLE 19 Comparison of Activities of UbiX Homologs in an In VitroAssay

An in vitro assay was followed to compare the activities of UbiX andfour of its homologs. In this in vitro assay the lysate from an E. colistrain over expressing AroY protein was combined with the lysate fromanother E. coli strain expressing UbiX protein or it homolog and thecombined lysate was assayed for its ability to consume PCA as asubstrate. In the muconic acid producing E. coli strain PCA isdecarboxylated by AroY protein to yield catechol which in turn isconverted into muconic acid by CatA protein. The decarboxylationactivity of AroY protein is expected to be enhanced by the presence ofUbiX or one of tis homolog and depending on the efficiency of UbiX orits homologs, the PCA in the assay solution will be consumed atdifferent rate.

In this in vitro assay UbiX and four of its homologs namely KpdB codedby kpdB gene of Klebsiella pnemoniae (kpdB) (SEQ ID 42), Elw coded bythe elw gene of E. coli W (SEQ ID 46), Kox coded by the kox gene ofKlebsiella oxytoca (kox) (SEQ ID 48) and Lpl coded by lpl gene ofLactobacillus plantarum (lpl) (SEQ ID 50). The names for the last threehomologs are simply provisional names given for this disclosure. AroY,UbiX, KpdB, Elw, Kox and Lpl were expressed from the strong constitutiveLambda Phage promoter P_(R) (SEQ ID3) on a low copy plasmid (SC101origin of replication). The plasmids pCAT350 (SEQ ID 55) and pCP165 (SEQID 56) were used for gene cloning. In constructing AroY plasmid, theprimers RP712 and RP714 were used to amplify the aroY gene and theprimers MS461 and MS346 were used to amplify the pCAT350 plasmidbackbone. The resulting PCR products were ligated to obtain a plasmidoverexpressing the AroY protein. In constructing a KpdB plasmid, theprimers RP731 and RP732 were used to amplify kpdB gene and the primersMS461 and MS346 were used to amplify the pCAT350 plasmid backbone. Theresulting PCR products were ligated to obtain a plasmid overexpressingKpdB protein. In constructing a UbiX plasmid, the primers MS669 andMS666 were used to amplify ubiX gene and the primers MS461 and RP607were used to amplify the pCAT350 plasmid backbone. The resulting PCRproducts were ligated to obtain a plasmid overexpressing UbiX protein.In constructing an Elw plasmid, the primers MS676 and MS680 were used toamplify elw gene and the primers MS461 and MS621 were used to amplifythe pCP165 plasmid backbone. The resulting PCR products were ligated toobtain a plasmid overexpressing the Elw protein. In constructing a Koxplasmid, the primers MS686 and MS684 were used to amplify the kox geneand the primers MS461 and MS621 were used to amplify the pCP165 plasmidbackbone. The resulting PCR products were ligated to obtain a plasmidoverexpressing Kox protein. In constructing an Lpl plasmid, the primersMS692 and MS691 were used to amplify the lpl gene and the primers MS461and MS621 were used to amplify the pCP165 plasmid backbone. Theresulting PCR products were ligated to obtain a plasmid overexpressingLpl protein. All fragments contained 20 bp of homology to enable cloningusing the NEBuilder HiFi DNA Assembly Cloning Kit and cloned into NEB5αE. coli cells (New England Biolabs).

Following plasmid cloning, an in vitro enzyme assay was developed todemonstrate AroY activity. 1 mL of an overnight LB grown culture wasspun down and resuspended in 200 μL of Bacterial Protein ExtractionReagent (B-PER) (Thermo Fisher Scientific). After 5 minutes incubationin a rotary mixer, the cell debris was removed by centrifuging thesamples at 13,000 rpm in a table top centrifuge. The clarified crudelysate supernatant was transferred to a new tube and stored on ice. 20μL of an AroY overexpression lysate was combined with 20 μL of UbiX or ahomolog lysate into a 150 μL reaction (final volume) containing 100 mMsodium phosphate buffer pH 6.4, 25 mM MgCl₂ and 1 mM protocatechuic acid(Sigma-Aldrich). The absorbance at 290 nm was read every minute for 60minutes. The AroY activity was measured by monitoring disappearance ofPCA at A290. The relative activities of the UbiX and its homologs areshown in FIG. 21. All UbiX homologs tested improved AroY activity, but awide variation of enzyme activity was produced depending on the specifichomolog tested. The highest AroY activity was achieved using KpdB, whilethe lowest activity was observed from the Lactobacillus homolog. Thewide range of activities shown can be employed to improve muconicpathway performance, as the highest activity may not always be optimal.

EXAMPLE 20 Effect of kpdB Expression Level on the Activity of AroY

Having established that that the expression of KpdB protein enhance theactivity of AroY protein in the in vitro assay, efforts were made todetermine if the level of expression of KpdB protein within the muconicacid producing biocatalysts would affect the level of muconic acidproduction. In this experiment, the muconic acid production strainMYR1305 was transformed with various plasmids that express KpdB atdifferent levels. Transformation of MYR1305 was conducted with threedifferent plasmids. In the experimental control, MYR1305 was transformedwith the control plasmid pCP140 without any genes coding for KpdBprotein. The DNA sequence of pCP140 is given in SEQ ID 57. Briefly,pCP140 was constructed to express an E. coli codon-optimized catAX underthe P15 promoter, E. coli tktA under native E. coli promoter, E. coliaroB under the P15 promoter, and E. coli aroD under P26 promoter (SEQ ID2). The second plasmid pCP169 used to transform MYR1305 is a derivativeof pCP140 additionally having the kpdB gene under the P26 promoter. Thethird plasmid pCP170 used to transform MYR1305 is a derivative of pCP140additionally having kpdB under the E. coli pgi promoter (SEQ ID 52). Lowlevel expression was achieved using the P26 promoter while highexpression was achieved using the E. coli pgi promoter. pCP169 andpCP170 were constructed by first amplifying the pCP140 plasmid in twofragments using two sets of primers (PCR primers RP607 and RP677 for thefirst fragment and the PCR primers RP671 and RP664 for the secondfragment). Two smaller PCR products facilitate easier plasmidconstruction than a single large PCR product. In constructing theplasmid pCP169, the P26 promoter was amplified using primers RP702 andRP783 and kpdB was amplified using primers RP781 and RP780. Inconstructing the plasmid pCP170, the pgi promoter was amplified usingprimers RP700 and RP784 and kpdB was amplified using primers RP779 andRP780. All PCR products had 20 bp homology overlaps to enable cloningusing the NEBuilder HiFi DNA Assembly Cloning Kit. As shown in FIG. 22,strains expressing kpdB produced higher levels of muconic acid than didthe control strains without any exogenous kpdB gene expression.Additionally, the PCA accumulation was eliminated in the strainsexpressing exogenous kpdB gene. The level of muconic acid produced didnot increase with the increased expression of kpdB gene suggesting asaturating level of activity was achieved even when the exogenous kpdBgene is expressed under low level of expression.

EXAMPLE 21 Complementation of a ppc Mutant and Effect on MuconicFormation

The increased availability of PEP for muconic acid formation wasinvestigated using the bacterial strains with the deletion of thephosphoenolpyruvate carboxylase (ppc) gene. The E. coli strain MYR1674was genetically engineered to use as a biocatalyst for muconioc acidproduction. MYR1674 is able to grow in minimal medium containing glucoseas a source of carbon and energy and produce muconic acid. However, whenthe ppc gene is deleted from MYR1674, the resulting stain MYR1674 Δppcis not able to grow in minimal medium containing glucose and is viableonly on rich media such as Luria Broth (LB). The loss of the ability ofMYR1674 Δppc to grow on minimal medium can be regained by means ofinserting the pyc gene coding for pyruvate carboxylase at the originalppc locus in the MYR1674 Δppc strain of E. coli.

The pyruvate carboxylase (pyc) gene from Saccharomyces cerevisiae (SEQID 53) was cloned using primers MS1383 and MS1384, containing flankinghomology to the E. coli ppc promoter and terminator. In order tofacilitate strong growth on minimal media, the strong constitutiverightward Lambda Phage promoter P_(R) was required. The P_(R) promoterwas amplified using primers MS1429 and MS1430 and the resulting PCRproduct was used to replace the endogenous ppc promoter. The finalnucleotide sequence for the Δppc:: P_(R)-pyc locus is shown in SEQ ID58. The S. cerevisiae pyc gene was chosen because it is not closelyrelated to any E. coli gene, and the expected lower expression due todiffering codon usage could be beneficial in preserving PEP for muconicacid production. There are many organisms that contain pyruvatecarboxylase, and any homologs or analogs having pyruvate carboxylaseactivity could be used. New strain MYR1772 derived from MYR1674 byintegrating Δppc:: P_(R)-pyc at the original ppc locus was viable on aminimal medium confirming the functionality of the cloned S. cerevisiaepyc gene. MYR1772 and its parent MYR1674 strains were compared in shakeflask experiments for their ability to produce muconic acid. As theresults shown in FIG. 23 indicates, MYR1772 strain produced higher titerof muconic acid production than parent MYR1674, demonstrating theadvantage in replacing the endogenous phosphoenolpyruvate carboxylaseenzyme with the exogenous pyruvate carboxylase enzyme.

EXAMPLE 22 Measuring Muconic Acid Production

The bacterial strains MYR814, MYR993, MYR1536, MYR1557, MYR1570,MYR1595, MYR1630, MYR1674 and MYR1772 were grown in shake flask culturesovernight and titer and yield for muconic acid production weredetermined. In addition, the growth rate was determined by measuring theabsorbance at 600 nm and the relative growth of various bacterialstrains are provided in Table 8. The bacterial growth represented by“+++” indicates a growth similar to the growth seen in a wild type Kcoil strain. The bacterial growth represented by “+” indicates poorgrowth. An intermediate growth is represented by “++”. When a particularstrain is showing a poor growth, that strain is subjected to 5 overnighttransfers to improve the growth, each transfer produces approximately 10generations or doublings.

The bacterial strains MYR814, MYR1570, MYR1630 and MYR1674 were grown in7 liter fermenters in a fed-batch mode and the titer and yield formuconic acid production were determined (Table 9). The bacterial strainstested for muconic acid titer and yield produced very little byproducts.For example, the bacterial strain after 72 hours of fed batchfermentation showed only 0.08 g/L of PCA and 0.07 g/L of fumarate asbyproducts.

TABLE 1 Bacterial strains used in the present invention Bacterial strainGenotype/Description ATCC8739 Escherichia coli “C” wild type MYR34ATCC8739 ΔaroE MYR170 ATCC8739 ΔaroE, ΔackA::P₁₅aroB MYR261 ATCC8739ΔaroE, ΔackA::P₁₅aroB, ΔpoxB::tktA MYR305 ATCC8739 ΔaroE,ΔackA::P₁₅aroB, ΔpoxB MYR31 ATCC8739 ΔptsHI, ΔgalP MYR217 ATCC8739ΔptsHI, ΔgalP, Δtdc::glf-glk, ΔaroE MYR352 ATCC8739 ΔaroE,ΔackA::P₁₅aroB, ΔadhE:: P₁₅-catAX, P_(R)-aroY, P₂₆-quiC RY903 ΔaroE,ΔackA::P₁₅aroB, pMG37, aroG*20-893 RY909 ΔaroE, ΔackA::P₁₅aroB, pMG37,aroG*20-899 RY911 ΔaroE, ΔackA::P₁₅aroB, pMG37, aroG*20-901 RY912 ΔaroE,ΔackA::P₁₅aroB, pMG37, aroGwt RY913 ΔaroE, ΔackA::P₁₅aroB, pMG37,aroG*20-893, ΔtyrR::kan RY919 ΔaroE, ΔackA::P₁₅aroB, pMG37, aroG*20-899,ΔtyrR::kan RY921 ΔaroE, ΔackA::P₁₅aroB, pMG37, aroG*20-901, ΔtyrR::kanRY922 ΔaroE, ΔackA::P₁₅aroB, pMG37, aroGwt, ΔtyrR::kan

TABLE 2 Bacterial used in the present invention Strain NameGenotype/Description Parent MYR802 ΔackA::P15-aroB ΔadhE::P15-catAX +P_(R)-aroY + P26-quiC ΔpflB::P15-catAX ΔtyrR aroG^(FBR) ΔpoxB:tktAΔptsHI Δtdc::P26-glf-glk ΔgalP aroE G105M MYR814 ΔackA::P15-aroBΔadhE::P15-catAX + P_(R)-aroY + P26-quiC ΔpflB::P15-catAX ΔtyrR MYR802aroG^(FBR) ΔpoxB::tktA ΔptsHI Δtdc::P26-glf-glk ΔgalP aroE G105M[P15-catA-catX + tktA + P15-aroB + P26-aroD in pCL1921 backbone] MYR993ΔackA::P15-aroB ΔadhE::P_(R)-aroY + P26-quiC ΔpflB::P_(R)-catAX ΔtyrRaroG^(FBR) MYR802 ΔpoxB::tktA ΔptsHI Δtdc::P26-glf-glk ΔgalP aroE G105MMYR993 ΔackA::P15-aroB ΔadhE::P_(R)-aroY + P26-quiC ΔpflB::P_(R)-catAXΔtyrR aroG^(FBR) MYR993 ΔubiX ΔpoxB::tktA ΔptsHI Δtdc::P26-glf-glk ΔgalParoE G105M ΔubiX::Kan^(R) MYR993 ΔackA::P15-aroB ΔadhE::P_(R)-aroY +P26-quiC ΔpflB::P_(R)-catAX ΔtyrR aroG^(FBR) MYR993 ΔubiD ΔpoxB::tktAΔptsHI Δtdc::P26-glf-glk ΔgalP aroE G105M ΔubiD::Kan^(R) MYR1305ΔackA::P15-aroB ΔadhE::P15-catAX + P_(R)-aroY + P26-quiCΔpflB::P15-catAX ΔtyrR MYR802 aroG^(FBR) ΔpoxB::tktA ΔptsHIΔtdc::P26-glf-glk ΔgalP ΔaroE Δzwf MYR1305 ΔackA::P15-aroBΔadhE::P15-catAX + P_(R)-aroY + P26-quiC ΔpflB::P15-catAX ΔtyrR MYR1305pCP140 aroG^(FBR) ΔpoxB::tktA ΔptsHI Δtdc::P26-glf-glk ΔgalP ΔaroE Δzwf[P_(R)-catA-catX + tktA + P15-aroB + P26-aroD in pCL1921 backbone]MYR1305 ΔackA::P15-aroB ΔadhE::P15-catAX + P_(R)-aroY + P26-quiCΔpflB::P15-catAX ΔtyrR MYR1305 pCP169 aroG^(FBR) ΔpoxB::tktA ΔptsHIΔtdc::P26-glf-glk ΔgalP ΔaroE Δzwf [P_(R)-catA-catX + tktA + P15-aroB +P26-aroD + P26-kpdB in pCL1921 backbone] MYR1305 ΔackA::P15-aroBΔadhE::P15-catAX + P_(R)-aroY + P26-quiC ΔpflB::P15-catAX ΔtyrR MYR1305pCP170 aroG^(FBR) ΔpoxB::tktA ΔptsHI Δtdc::P26-glf-glk ΔgalP ΔaroE Δzwf[P_(R)-catA-catX + tktA + P15-aroB + P26-aroD + P_(PG1)-kpdB in pCL1921backbone] MYR1536 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrR aroG^(FBR)ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR802 ΔgalP::P15-ubiX ΔpflB::P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI:: P_(R)-aroYΔ0039:: P_(R)-catAX MYR1557 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrRaroG^(FBR) ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR1536 ΔgalP::P15-ubiXΔpflB:: P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI::P_(R)-aroY Δ0039:: P_(R)-catAX (Evolved version of MYR1536 for fastergrowth) MYR1570 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrR aroG^(FBR)ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR1536 ΔgalP::P15-ubiX ΔpflB::P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI:: P_(R)-aroYΔ0039:: P_(R)-catAX Δzwf (Evolved version of MYR1536 for faster growth)MYR1595 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrR aroG^(FBR)ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR1557 ΔgalP::P15-ubiX ΔpflB::P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI::P_(R)-AroY Δ0039::P_(R)-catAX Δzwf ΔpykF MYR1630 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrRaroG^(FBR) ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR1595 ΔgalP::P15-ubiXΔpflB:: P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI::P_(R)-aroY Δ0039:: P_(R)-catAX Δzwf ΔpykF Δ2160:: P_(R)-aroG^(FBR)MYR1674 ΔackA::P_(acpp)-aroB ΔadhE::P15-qa4 ΔtyrR aroG^(FBR)ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glk MYR1630 ΔgalP::P15-ubiX ΔpflB::P_(R)-CatAX P_(acpp)-aroD ΔmgsA::P_(rpIU)-qa4 ΔptsHI:: P_(R)-aroYΔ0039:: P_(R)-catAX Δzwf ΔpykF Δ2160:: P_(R)-aroG^(FBR) (Evolved versionof MYR1630 for faster growth) MYR1772 ΔackA::P_(acpp)-aroBΔadhE::P15-qa4 ΔtyrR aroG^(FBR) ΔpoxB::P_(R)-tktA Δtdc:: P26-glf-glkMYR1674 ΔgalP::P15-ubiX ΔpflB:: P_(R)-CatAX P_(acpp)-aroDΔmgsA::P_(rpIU)-qa4 ΔptsHI:: P_(R)-aroY Δ0039:: P_(R)-catAX Δzwf ΔpykFΔ2160:: P_(R)-aroG^(FBR) Δppc:: P_(R)-pyc

TABLE 3 Plasmids used in the present invention Bacterial PlasmidGenotype/Description YEp24 2μ yeast origin, URA3, Tc^(R), pMB1 replicon,Ap^(R) pCP32AMP 2μ yeast origin, URA3, Tc^(R), pMB1 replicon, Ap^(R),aroG pCP14 2μ yeast origin, URA3, Tc^(R), pMB1 replicon, Ap^(R), P₁₅aroBpCP54 2μ yeast origin, URA3, Tc^(R), pMB1 replicon, Ap^(R), P₁₅aroB,aroG pCP50 2μ yeast origin, URA3, Tc^(R), pMB1 replicon, Ap^(R), aroG,tktA pCP55 2μ yeast origin, URA3, Tc^(R), pMB1 replicon, Ap^(R), aroG,aroB, tktA pCL1921 pSC101 replicon, Spc^(R) pMG27 pSC101 replicon,Spc^(R), P_(R)-aroY pMG31 pSC101 replicon, Spc^(R), P₁₅-catAX pMG33pSC101 replicon, Spc^(R), P₁₅-catAX , P_(R)-aroY pMG37 pSC101 replicon,Spc^(R), P₁₅-catA-CatX, P_(R)-aroY, P₂₆-quiC pMG39 pSC101 replicon,Spc^(R), P₂₆-quiC pMG47 pSC101 replicon, Spc^(R), P₁₅-catAX, P_(R)-aroY,P₂₆-asbF pMG70 pSC101 replicon, Spc^(R), P₁₅-catAX, P_(R)-aroY, P₂₆-qa-4

TABLE 4 aroG*mutant alleles that lead to resistance to phenylalaninefeedback inhibition Allele Nucleotide Amino acid Strain number mutationmutation RY893 aroG*20-893 C449T Pro150Leu RY897 aroG*20-897 C449TPro150Leu RY899 aroG*20-899 T538C Ser180Pro RY901 aroG*20-901 C438TPro150Ser MYR450 aroG*111 C55T Pro19Ser MYR451 aroG*211 G533A Gly178GluMYR452 aroG*212 C540T Ser180Phe MYR453 aroG*311 Deletion from baseDeletion of pair 36 to 44bp Glu-Ile-Lys MYR454 aroG*312 C632T Ser211PheMYR455 aroG*411 T29A Ile10Asn MYR456 aroG*412 G533A Gly178Glu MYR457aroG*511 C448T Pro150Ser

TABLE 5 AroG activity measurement in crude extract from variousrecombinant E. coli strains Specific activity mU (One mU = one nMproduct made per % of activity milligram protein per resistant to StrainaroG* allele minute) phenylalanine RY893 aroG*20-893 62 34 RY897aroG*20-897 55 77 RY899 aroG*20-899 92 113 RY901 aroG*20-901 78 76 RY902aroG wild type 38 6 RY890 aroG wild type 54 7

TABLE 6 Muconic acid production in shake flasks by strains containingfeedback resistant aroG* alleles Strain Muconic acid titer g/l RY9133.04 RY919 3.11 RY921 2.99 RY922 1.45

TABLE 7 Sequence Information No. Name Description  1 SEQ ID No. 1The P₁₅ promoter from Bacillus subtilis phage SP01, with a stem and loopadded just downstream from the transcription start site.  2 SEQ ID No. 2The P₂₆ promoter from Bacillus subtilis phage SPO1  3 SEQ ID No. 3The P_(R )promoter from Escherichia coli phage  4 SEQ ID No. 4Protein sequence of 3-dehydroshikimate dehydratase from Neurospora crassaencoded by the qa-4 gene.  5 SEQ ID No. 5Genomic DNA sequence of the qa-4 gene from Neurospora crassa plussurrounding sequences.  6 SEQ ID No. 6Protein sequence of 3dehydroshikimate dehydratase from Aspergillusnidulans. encoded by the qutC gene  7 SEQ ID No. 7Genomic DNA sequence of the qutC gene from Aspergillus nidulans plussurrounding sequences  8 SEQ ID No. 8Protein sequence of protocatechuate decarboxylase (AroY) from Klebsiellapnemoniae ATCC25597  9 SEQ ID No. 9DNA sequence of the aroY gene of Klebsiella pneumoniae 342 plus 2kilobases of surrounding DNA sequences 10 SEQ ID No. 10DNA sequence of the catA gene from Acinetobacter baylyi ADP1, includingupstream sequence and two open reading frames downstream410 bases of  11SEQ ID No. 11Protein sequence of CatA (catechol 1,2-dioxygenase) from Acinetobacterbaylyi ADP1 12 SEQ ID No. 12DNA sequence of the quiC (3-dehydroshikimate dehydratase)gene fromAcinetobacter sp. ADP1 13 SEQ ID No. 13Codon-optimized DNA sequence of the quiC (3-dehydroshikimatedehydratase)gene from Acinetobacter sp. ADP1 14 SEQ ID No. 14Protein sequence of QuiC (3-dehydroshikimate dehydrogenase fromAcinetobacter sp. ADP1 15 SEQ ID No. 15DNA sequence of the plasmid pAC21 16 SEQ ID No. 16DNA sequence of the plasmid pAC19 17 SEQ ID No. 17DNA sequence of the plasmid pMH17F 18 SEQ ID No. 18DNA sequence of the coding region of the wild type aroG gene 19SEQ ID No. 19 DNA sequence of the plasmid pMH28F 20 SEQ ID No. 20DNA sequence of the plasmid pCL1921 21 SEQ ID No. 21DNA sequence of the plasmid pMG27 22 SEQ ID No. 22DNA sequence of the plasmid pMG31 23 SEQ ID No. 23DNA sequence of the plasmid pMG33 24 SEQ ID No. 24DNA sequence of the plasmid pMG37 25 SEQ ID No. 25DNA sequence of the plasmid pMG39 26 SEQ ID No. 26DNA sequence of the plasmid pMG47 27 SEQ ID No. 27DNA sequence of the plasmid pMG70 28 SEQ ID No. 28DNA sequence of the plasmid pCP32AMP 29 SEQ ID No. 29DNA sequence of the plasmid pCP14 30 SEQ ID No. 30DNA sequence of the plasmid pCP50 31 SEQ ID No. 31DNA sequence of the plasmid pCP54 32 SEQ ID No. 32DNA sequence of the plasmid pCP55 33 SEQ ID No. 33DNA sequence of the plasmid YEP24 34 SEQ ID No. 34DNA sequence of the deleted aroE region 35 SEQ ID No. 35DNA sequence of the integrated cassette Δack::P₁₅aroB 36 SEQ ID No. 36DNA sequence of the ΔpoxB region 37 SEQ ID No. 37DNA sequence of the integrated cassette ΔpoxB::iktA 38 SEQ ID No. 38DNA sequence of the ΔptsHI region 39 SEQ ID No. 9DNA sequence of the integrated cassette Δtdc::glf-glk 40 SEQ ID No. 40DNA sequence of the ΔgalP region 41 SEQ ID No. 41MYR352 ΔadhE::P₁₅-catAX, P_(R)-aroY, P₂₆-quiC 42 SEQ ID No. 42Nucleotide sequence of Klebsiella pneumoniae kpdB gene. 43 SEQ ID No. 43Amino acid sequence of Kpd13 protein of Kiebsiella pneumoniae 44SEQ ID No. 44 Nucleotide sequence of Escherichia coli ubiX gene 45SEQ ID No. 45 Amino acid sequence of UbiX protein of Escherichia coli.46 SEQ ID No. 46Nucleotide sequence of Escherichia coli W strain elw gene 47SEQ ID No. 47Amino acid sequence of Elw protein of Escherichia coli W strain. 48SEQ ID No. 48 Nucleotide sequence of Klebsiella oxytoca kox gene 49SEQ ID No. 49 Amino acid sequence of Kox protein of Klebsiella oxytoca.50 SEQ ID No. 50 Nucleotide sequence of Lactobacillus plantarum lpl gene51 SEQ ID No. 51Amino acid sequence of Lp1 protein of Lactobacillus plantarum. 52SEQ ID No. 52 Nucleotide sequence of Pgi promoter 53 SEQ ID No. 53Nucleotide sequence of Saccharomyces cerevisiae pyc gene 54SEQ ID No. 54Amino acid sequence of Pyc protein of Saccharomyces cerevisiae. 55SEQ ID No. 55 DNA sequence of the plasmid pCAT350 56 SEQ ID No. 56DNA sequence of the plasmid pCP165 57 SEQ ID No. 57DNA sequence of the plasmid pCP140 58 SEQ ID No. 58Nucleotide sequence of Δppc::P_(R)-pyc 59 SEQ ID No. 59Nucleotide sequence of acpP promoter 60 SEQ ID No. 60Nuclotide sequence of rp1U promoter 61 MS604AACGCCGTATAATGGGCGCAGATTAAGAGGCTACAGTGGGCTTACATGGCGATAGCTAGA 62 MS605TGTCGGATCGATAAATAGGGCAAAACAAACGCGCATCCCGGAAAACGATTCCGAAGCCCA 63 MS608AAAGTCTGCCTGCAAGTCTGACAGGGCAACTATTTGTGGGCTTACATGGCGATAGCTAGA 64 MS609TTGCAAAATTGCCCTGAAACAGGGCAACAGCGGAGTCCCGGAAAACGATTCCGAAGCCCA 65 MS461GGCTATATTCCTTATCTAGATTAGT 66 MS346 GTCTGACAGGTGCCGGATTTCATAT 67 RP712TCTAGATAAGGAATATAGCCATGACCGCACCGATTCAGGATCTGC 68 RP714AAATCCGGCACCTGTCAGACTTATTTTGCGCTACCCTGGTTTTTT 69 RP731CATGTACTAATCTAGATAAGGAATATAGCCATGAAACTGATTATTGGGATGACGGGGGCC 70 RP732GCCGGATATGAAATCCGGCACCTGTCAGACTTATTCGATCTCCTGTGCAAATTGTTCTGC 71 MS669TCTAGATAAGGAATATAGCCATGAAACGACTCATTGTAGGCATCA 72 MS666ACCGAACAGGCTTATGTCCAGATAGCAGGTATAGCGGTTGAATCG 73 RP607TGGACATAAGCCTGTTCGGTTCGT 74 MS621 TTAGATTTGACTGAAATCGTACAGT 75 MS676TCTAGATAAGGAATATAGCCATGAAACTGATCGTCGGGATGACAG 76 MS680ACGATTTCAGTCAAATCTAATTATTCATTCTCCTGAGAAAAATTC 77 MS686TCTAGATAAGGAATATAGCCATGACGGCACGCATCATCATTGGTA 78 MS684ACGATTTCAGTCAAATCTAATTAATTAAAACGTAGCTCGCCTTCA 79 MS692TCTAGATAAGGAATATAGCCATGAAACGAATTGTTGTGGGAATCA 80 MS691ACGATTTCAGTCAAATCTAATTAATCCCCCTCCCAACGGCGATCA 81 RP677CGACGTTGTAAAACGACGGCCAGTG 82 RP671 TTAATCGCCTTGCAGCACATCCCCC 83 RP664ACGAACCGAACAGGCTTATGTCCA 84 RP702GCCGTCGTTTTACAACGTCGGATCCGCCTACCTAGCTTCCAAGAA 85 RP783CCTACAATGAGTCGTTTCATTAGGTTTTCCTCAACCCGGGAGCGT 86 RP781CCCGGGTTGAGGAAAACCTAATGAAACGACTCATTGTAGGCATCA 87 RP780ATGTGCTGCAAGGCGATTAAGATAGCAGGTATAGCGGTTGAATCG 88 RP700GCCGTCGTTTTACAACGTCGAGCGGGGCGGTTGTCAACGATGGGG 89 RP784CCTACAATGAGTCGTTTCATGGCTATATTCCTCCTCTGCATGAGA 90 RP779TGCAGAGGAGGAATATAGCCATGAAACGACTCATTGTAGGCATCA 91 MS1383GATGGGGTGTCTGGGGTAATATGTCGCAAAGAAAATTCGCCGGCT 92 MS1384GGGTTTGCAGAAGAGGAAGATCATGCCTTAGTTTCAACAGGAACT 93 MS1429ATTCCTGCTATTTATTCGTTCGTTAAATCTATCACCGCAAGGGAT 94 MS1430GCGAATTTTCTTTGCGACATGGCTATATTCCTTATCTAGATTAGT

TABLE 8 Titer and yield for muconic acid production and growth ofvarious E. coli strains in shake flask cultures Titer Yield (gramsStrain (grams muconic muconic acid/ Name acid/liter) gram glucose)Growth MYR814 2 0.16 +++ MYR993 1.9 0.15 +++ MYR1536 2.7 0.22 + MYR15573 0.25 +++ MYR1570 4.5 0.38 ++ MYR1595 4.5 0.38 ++ MYR1630 4.8 0.4 +MYR1674 3.8 0.32 +++ MYR1772 5 0.42 +++

TABLE 9 Titer and yield for muconic acid production for various E. colistrains in fed batch cultures Titer Yield (grams Strain (grams muconicmuconic acid/ Name acid/liter) gram glucose) Time (hours) MYR814 30.90.20 48 MYR1570 49.0 0.36 48 MYR1630 58.3 0.47 48 MYR1630 69.5 0.42 72MYR1674 81.5 0.43 72

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1-30. (canceled)
 31. A genetically engineered microorganism thatproduces muconic acid from a non-aromatic carbon source comprising atleast one exogenous gene encoding for 3,4-dihydroxybenzoic aciddecarboxylase and at least one gene that codes for a protein thatincreases the activity of said 3,4-dihydroxybenzoic acid decarboxylase.32. The genetically engineered microorganism of claim 31, wherein saidgenetically engineered microorganism produces at least 60 g/L of muconicacid in 72 hours.
 33. The genetically engineered microorganism of claim31, wherein said genetically engineered microorganism is a bacterium.34. The genetically engineered microorganism of claim 31, wherein saidgenetically engineered microorganism is Escherichia coli.
 35. Thegenetically engineered microorganism of claim 31, wherein said proteinthat increases the activity of said 3,4-dihydroxybenzoic aciddecarboxylase is a KpdB protein encoded by a kpdB gene or a homologthereof, and wherein said KpdB protein or homolog thereof has at least25% amino acid identity to said KpdB protein.
 36. The geneticallyengineered microorganism of claim 31, wherein said protein thatincreases the activity of said 3,4-dihydroxybenzoic acid decarboxylaseis a UbiX protein encoded by a ubiX gene or a homolog thereof, andwherein said UbiX protein or homolog thereof has at least 25% amino acididentity to said UbiX protein.
 37. The genetically engineeredmicroorganism of claim 31, further comprising one or more exogenousgenes selected from the group consisting of aroZ, qa-4, asbF, quiC andcatAX.
 38. The genetically engineered microorganism of claim 31, furthercomprising one or more exogenous genes selected from the groupconsisting of aroB, aroD, aroF, aroG, aroH, tktA, talB, rpe, and rpi,wherein said one or more exogenous genes encode one or more proteinsthat function in a shikimic acid pathway.
 39. The genetically engineeredmicroorganism of claim 31, further comprising at least one exogenousgene encoding a pyruvate carboxylase and a mutation of the phosphoenolpyruvate carboxylase gene.
 40. A genetically engineered Escherichia colistrain that produces muconic acid from a non-aromatic carbon sourcecomprising at least one exogenous gene encoding for 3,4-dihydroxybenzoicacid decarboxylase and at least one gene that codes for a protein thatincreases the activity of said 3,4-dihydroxybenzoic acid decarboxylase,wherein said genetically engineered Escherichia coli strain produces atleast 60 g/L of muconic acid in 72 hours.
 41. A genetically engineeredEscherichia coli strain that produces muconic acid from a non-aromaticcarbon source comprising at least one exogenous gene encoding for3,4-dihydroxybenzoic acid decarboxylase and at least one gene that codesfor a protein that increases the activity of said 3,4-dihydroxybenzoicacid decarboxylase, wherein said genetically engineered Escherichia colistrain produces at least 80 g/L of muconic acid in 72 hours.
 42. Thegenetically engineered Escherichia coli strain of claim 40, wherein saidprotein that increases the activity of said 3,4-dihydroxybenzoic aciddecarboxylase is a KpdB protein encoded by a kpdB gene or a homologthereof, and wherein said KpdB protein or homolog thereof has at least25% amino acid identity to said KpdB protein.
 43. The geneticallyengineered Escherichia coli strain of claim 40, wherein said proteinthat increases the activity of said 3,4-dihydroxybenzoic aciddecarboxylase is a UbiX protein encoded by a ubiX gene or a homologthereof, and wherein said UbiX protein or homolog thereof has at least25% amino acid identity to said UbiX protein.
 44. The geneticallyengineered Escherichia coli strain of claim 40, further comprising oneor more exogenous genes selected from the group consisting of aroZ,qa-4, asbF, quiC and catAX.
 45. The genetically engineered Escherichiacoli strain of claim 40, further comprising at least one exogenous geneencoding a pyruvate carboxylase and a mutation of the phosphoenolpyruvate carboxylase gene.